 Okay, well welcome back and I think that starting out today we should probably decide on a review session. Okay, so how many people, we have to do it this week because unfortunately next week the room's not available. And so how many people would like to do it on Wednesday at this time? Okay, so we need to hold a review session or I'd like to. It's not obligatory but it would be nice to hold a review session and if we did it on, we have to either Wednesday or Friday this week. So how many for Wednesday again? Okay, how about Friday? Looks like it's Wednesday then so we'll meet here on Wednesday. And I'll continue to hold office hours up to the day of the final. But next week I'll probably be, I'll arrive at the office and if nobody shows up within about, by about 20 after I'm probably going to leave because I don't know how many people will even be on campus next week. But so be sure if you come to office hour next week that you show up within the first 10 minutes like between 10 after and 20 after. And same goes for the day of the final. All right, so last time I gave you a bit of an introduction to the whole topic of signaling in plants, how plants can actually perceive stimuli. We talked about the stimulus of light and photoperidism in particular that is physiological responses of plants to the relative length of light and darkness. Which is important with regard to flowering in particular is one of the things we mainly focused on. I just want to finish up on that by pointing out that it's actually not the length of the day that's important but it's the uninterrupted length of the night that plants are actually perceiving. And this was established after the name short day plant and long day plant were already ingrained in the literature. So those names persist for plants that flower during short days and plants that flower during long days. And as I mentioned, there's some plants that are day-neutral, they just flower whenever they're at the physiological state to flower. But the experiments that were done to demonstrate that this is a night-length response rather than a day-length response were pretty elegant. Just basically growing plants in a greenhouse situation under controlled light. And with the short day plant, which would normally flower after the day length went below a certain level in the fall or the late summer, a brief flash of light was introduced about midway through the dark period and those plants then would flower. And the same sort of thing was observed with the long day plants which typically would flower when day-length reaches a certain period of time that flower in the late spring or early summer. And it turned out that a brief flash of intense light, just the briefest flash in both cases, would initiate flowering even though the dark period was too long still for flowering to occur in this case. So it appears that based on other work that Phytochrome actually is involved here at least in part as one of the photoreceptors sensing the overall length of the night period. And with the flash of light Phytochrome signals the end of that night period. So with the long day plant, which is basically a short night plant, that flash of light breaks up that long dark period into two short periods and the plant senses it as a short night and flowering is initiated. And here where the flash of light was also introduced in the middle of that dark period. I actually misstated this, that the short day plant will not flower because the long night is not perceived, instead it's perceived as a short night. So these plants flower when the night length crosses a critical threshold, but the flash of light causes the plant not to perceive that as a long night. Whereas here the flash of light suggests this is a short night, which is what it's required for a long day plant to flower. Any questions about that then? So this is all really have to say about, well there's one more thing to say about photoperitism. It's pretty interesting and this segues into the next topic about growth regulation. And that is that experiments were also done where a plant that was, where flowering had been initiated, in this case a short day plant, had a long day plant grafted onto it. So physically grafted onto the tissues of this short day plant. This is a long day plant that hadn't flowered at the time. And this plant then flowered even though the day length wasn't appropriate for a long day plant to flower under those conditions. So this kind of experiment could be done the other way around too, with grafting a short day shoot onto a long day plant that had already initiated. And it would flower at a time when it normally wouldn't. So clearly something is being transmitted through the tissues of this plant that's undergone flowering to the tissues of this other plant that's resulting in initiation of flowering. And this sort of experiment dates long ago. For nearly three quarters of a century people have oscillated on the basis of such data that there is a flowering hormone that's transmitted within plants. They've even given it the name Florogen, but no one has ever successfully isolated this hormone so it's still a complete hypothetical if it really exists. But it's still called by this name in the literature and people discuss it as if it exists. It's one of the stranger aspects of plant physiology. And it's possible based on some recent experiments in Arabidopsis, which is a model system for studying plant biology, that the stimulus might not be a hormone but might be a macromolecule protein or a nucleic acid rather than a micromolecule, which hormones typically are. So that gets to the point of plant chemistry. And I want to talk about plant chemistry only in relation to how plants respond to environmental stimuli. And this is a really fascinating area about which we're learning more all the time. Huge amounts have been learned since I actually took botany and taught it as a graduate student. But plants like animals have a lot of different ways for coping with environmental change. And of course most animals can move in response to either a favorable stimulus or an unfavorable stimulus. They can either approach it or retreat from it. Whereas plants through most of their life cycle are immobile and are locked in place essentially. So their responses really are typically in the area of growth or metabolic changes. And plant hormones are one of the ways that the plant can signal this necessity to change to undergo some kind of response. And these are often called plant growth regulators because they differ from animal hormones in some ways. They sometimes act close to the source of production. But they're typically translocated within the plant or are moved within the plant like an animal hormone. And like animal hormones they're organic compounds that modify or control one or more physiological responses. And those physiological responses often have to do with promoting or inhibiting growth. So these are responses like cell division, cell elongation or cellular differentiation or maturation. Three major aspects of growth. And I just mentioned this, they're often transported from the site of stimulus reception to the site of response. And one of the interesting things about hormones in general is that they can have a very strong effect with very low concentrations. And the amount of the hormone present can have a huge effect on its action on the signal that it provides to the plant. And also that action can be affected by the type of tissue in which the hormone is in contact or the age of that tissue, its state of maturation. And these are involved in just about every aspect of plant growth and development. And one of the interesting things about plant hormones that makes them so difficult to study but also fascinating is that an individual hormone can be involved in many different responses in plants. As I just mentioned, the site of action, the age of the tissue, the amount of hormone present can have a big effect on the response. And also an individual response can involve multiple hormones, different hormones. And it's the relative concentration of these different hormones that's most important generally what we can think of as hormonal balance, which is something we also think about in animals. Well, the hormonal balance talk about that a couple of times here today is really important. So just like so many things in biology, some of the earliest insightful work on plant hormones was done by Charles Darwin in association with his son Francis back near the end of his life in the 1880s. And what they did is they grew up grass seedlings. And remember grasses germinate directly through the soil. The tip of the shoot emerges sheathed in what's called a coley-optile, the sheath that emerges directly from the soil. And it was the bending of these coley-optiles in response to light that the Darwin studied. And so they found that these coley-optiles would bend toward the light, which is a pretty adaptive response it would seem in terms of maximizing photosynthesis. And so they wanted to understand how that occurred and what that involved. So they did various things, but one of the things they did that was interesting is they cut off the tip of that coley-optile. And they saw that no bending, this growth response that resulted in bending didn't occur. And so maybe that was just some sort of wound problem with wounding the plants. So they covered up the tip with a light impenetrable cover and they also saw no bending. So the next thing they tried is they covered up the area where the bending actually occurs and the things still bent. So they concluded that light is sensed by the top of the shoot and that some sort of hormonal signal is sent down through the plant to a point of response where there's a growth response that results in bending toward light. So this was the first evidence for plant hormones. And this elicited a lot of interest by others and there were other experiments done with the same system after Darwin. And one of them was this one, which established that this phototropic signal was actually a chemical as Darwin suspected. And what was done here was that the sliced-off tip of the coley-optile was placed on a little block of auger. We talked about auger, of course, as this carbohydrate extracted from red algae and it's permeable. This was placed in between the rest of the coley-optile and its tip. And what was seen is this growth response still occurred whereas if an impenetrable object was placed between the tip and the rest of the coley-optile, in this case a little sheet of mica, there was no bending. So the conclusion from this experiment was that indeed some chemical was transmitted down through the coley-optile as Darwin suspected. Okay, and then it wasn't until the 1920s that Fritz Went at Caltech performed a kind of decisive experiment here where basically using the same system, this tip of the coley-optile that had been initiated or had been exposed to light and had undergone some exposure to light was cut off and placed again on a little block of auger with a sufficient time for any diffusion to occur into this auger, which is shown here by the red, the active substance here. And then this was placed on different coley-optiles that had also had their tips excised that hadn't been exposed to light. And it was placed offset to one side, either one side or the other, on different coley-optiles. And a growth response was seen with bending away from the edge that the auger was placed on. And when the tissues of those coley-optiles was looked at in more detail down near the area of bending, Went saw that the cells had elongated along the side that the auger was placed on. So the idea here was that then not only was a chemical transmitted down here, the chemical was resulting in an elongation of cells along the side or in response to this chemical. And so apparently on the dark side of the coley-optile, away from light, there was a growth response by this transmitted chemical, which he called oxen, which just means to increase. It wasn't known what it was at the time. It was later discovered to be endolacetic acid. And there are some other related substances called oxens as well that have a number of different responses. And so this was the first plant hormone that was really studied. And it turns out to be a really important plant hormone. And like other plant hormones, it's involved in a number of different effects. And one of the ones we've already talked about is apical dominance. That is how a chute will have a primary apical meristem that's active, whereas axillary meristems will be suppressed, will be inhibited, or won't be active. And it turns out, as we've already mentioned, if you cut off the tip, the active tip of a chute, these axillary buds that are found in the upper axles of leaves here will suddenly be initiated. And here you can see a little some evidence of this. This tip was just cut off. And here go the axillary buds becoming new chutes that are growing upward like the original tip. And one typically becomes dominant. So that's pretty strong and clear response to the removal of the apical meristem. And it turns out that studies of the presence of oxen within chutes shows that the active apical meristem is where oxen is produced at relatively high concentrations. And with its removal then, we have removal of what appears to be an inhibitor of these side chutes, of these axillary buds. And we'll get more to that in a minute. So as I mentioned, also these plant hormones have different effects on different tissues. And another completely different effect occurs with oxen in the presence of ovary tissue. And so one of the things that's been seen is that oxen is produced in proximity to or perhaps by the seeds of a developing ovary, a developing fruit. And here we can see a normal tomato where there was normal pollination and fertilization with the oxen produced in the vicinity of the seeds being important for normal fruit development. And in the absence of fertilization of those seeds, if there's no pollination, the tomato fruit won't develop. But if you apply oxen, if you spray oxen on that ovary, even if there's no pollination, the ovary can develop. And this is done to ensure fruit development in tomatoes and some other fruits in the absence of any pollinators. So oxen does have this ability, this signaling for fruit development. So again, oxen is involved in cell elongation, not division, but elongation. And coley optiles on the dark side of the shoot, on stems, on the light side it turns out. So those experiments by Darwin and others were a little bit misleading. Oxen operates in slightly different ways in stems, but also in regard to elongation of cells. It also operates in apical dominance, fruit development, and in lateral root formation, formation of branching from the roots as we'll talk about in a moment. All right, so another point that I made earlier just briefly was that different concentrations of hormones can have really different effects on the tissues that they're in contact with. And this is the case with oxen. As you can see, here's a gradient of oxen from low to high concentration, higher in this direction. And here we can see a positive effect of growth effect and an inhibitory effect of oxen with no effect here. So what you see here is the different parts of the plant are just sensitive to different concentrations. But in each case, there's a response that's quite similar as oxen is increased, but at different levels. So we initially get an increase in growth, but then an inhibition of growth. And if we look at this in stems, you can see the concentration of oxen that's maximally stimulative of stem growth is strongly inhibiting of bud growth, of those axillary buds, as that experiment of cutting off the tip showed. And of course, the stem tip is producing a lot of oxen, so you cut off that tip, the amount of oxen is reduced in the stem. And we end up with maximal initiation of buds and suppression of roots at that level. Stems aren't even affected at the level that initiates bud growth in terms of the main stem. So the question? Yeah, Brad? That's right. Yeah, it's just a generic term. It's not specific as to chemistry particularly. That's right. Cytokinans were discovered much later in the 1940s, but they're also really important. And it turns out that the balance of cytokinin to oxen is really important for the plant growth response. And so cytokinans are named because they promote cell division. And cytokinin comes from cytokinesis or cell division. Okay, so that's an easy way to remember the term. And remember that oxen's promote elongation of cells, not division of cells. So cytokinans operating with oxen are important for the overall growth response since both division and elongation are important in growth. And so a high cytokinin to oxen ratio promotes activation of chute branching, this axillary bud branching that we talked about. And a low cytokinin to oxen ratio promotes activation of lateral roots, so branching of the roots. And I mentioned that oxen can stimulate lateral root production. And it's really the low cytokinin to oxen ratio that promotes that relatively high oxen compared to cytokinin here. So I already mentioned this, that cytokinin promotes development of branches. Actually I didn't mention that. I mentioned that the reduction in the amount of oxen results in the branching of the side chutes. But what it really is, is that the cytokinin ratio, there's a high cytokinin to oxen ratio after the tip is removed. And that's what's important with regard to the overall branching of the chute. And interestingly, the cytokinins and oxens are produced in different parts of the plant body and they're mobilized. They move in opposite directions in the plant. So the oxen is moving, as I mentioned, from the chute tips downward. And it still moves toward the roots even if you turn the plant upside down. This is not a gravity response. And cytokinins are typically produced in the roots and transported upward toward the chute. And so this creates a gradient of cytokinin and oxen within the plant body that's important in maintaining this hormonal balance across the plant. Okay, so that's one of the interesting things that was discovered about this interplay between cytokinins and oxens. Is their direction of movement is different, their place of production is different. And that helps to set up this gradient that's really important in growth. All right, so cytokinins then important in cell division and differentiation in concert with oxens involved in cell elongation for the overall growth response. And also axillary bud growth, particularly when apical dominance is removed by damage to the chute tip. But also, for example, at lower points on the stem where you'd have a higher cytokinin to oxen ratio, as you get further and further away from the chute tip, there's less inhibition of buds typically. All right, another class of, third class of plant hormones are called gibberellons. And these are called gibberellons because they were originally discovered in a plant that was infected by a fungus called gibberella. It's just an aside, but that's where the name comes from. And this fungus produced a compound that we call gibberellon that resulted in rice seedlings being strongly, being very spindly, having an uncontrolled growth response. And so gibberellons have a strong effect in stem elongation and overall have a strong growth stimulating effect. So these are substances that tend to have a strong growth stimulating effect. They're also important in promoting seed germination, so the breaking of dormancy in seeds, especially plants that have a light requirement for germination. And we use gibberellons all the time at really low concentrations to germinate seeds of certain plant species in my lab. And you can go from a plant that you can barely get to germinate. You have to dissect the embryos out to get them to germinate to having them almost jump out of the seeds. I mean, just within less than 12 hours, they'll be fully germinated. So these have a pretty strong effect in plants that recognize them as a stimulus for germination. And they're also used commercially to promote fruit enlargement and bunch length in seedless grapes. We already talked about how oxen can stimulate the initiation of growth, fruit development, and gibberellons can actually result in larger fruits. Here's a bunch of grapes that weren't treated with a gibberellon spray, and here are some that were treated with a gibberellon spray. So gibberellons have a lot of uses, and they also can be produced by plants as a wound response and can promote healing in response to wounding. They have a number of responses. We're just sort of scratching the surface here in terms of some of the more outstanding functions. And you've probably seen bolting occur before in herbs that just have basil leaves until they start to produce a flowering shoot. And the production of a flowering shoot can happen really quickly, so quickly that it's called bolting. And this is something that's stimulated by gibberellic acid as well. And when I actually was a GSI in a class like this many years ago, we didn't even know about these compounds, these bresino steroids. These have just been recently discovered to be plant hormones, and they're actually steroids similar to animal sex hormones in terms of their overall chemistry. But they're so similar in function to oxen in terms of promoting elongation of stems that they weren't recognized as distinct from oxen. And it's plants like these that allowed them to be recognized. These little dwarf ones here are ones that have minimal, inter-notal elongation and happen to be mutants that don't produce bresino steroids. And this name bresino steroids comes from the fact that these were discovered in Arabidopsis, which is the model system for plant biology. And it's in the Brassicae family or the Mustard family, just an aside, but that's what the name comes from. So just to let you know that these are still being discovered, this was a fairly recent discovery, similar to oxen. And this is a really important plant hormone, abscissic acid, we just call it ABA. This actually works antagonistically to gibberellins, and the relative balance of gibberellins and ABA can be important in the growth response. So I mentioned gibberellins have a really strongly stimulative effect to growth and seed germination. Whereas abscissic acid is just the opposite. It has an inhibiting effect on stem growth. And it's important in inducing seed dormancy and preparing the seed for desiccation as the seed matures. So dormancy in seeds is usually often responsible, or abscissic acid is often responsible for that. And it requires long periods of cold or some other stimulus to break down ABA and release the plant from that dormancy condition. Interestingly, there are some plants that don't produce much ABA in their seeds and don't have dormancy. Mangrows are really notable in this sense. They germinate while they're still on the parent plant. Here you can see the radicals, the embryonic roots emerging from seeds still in the fruits of a mangrove that haven't been dropped yet from the parent plant. And this kind of precocious germination is important for the survival of mangroves, and it just so happens that those seeds are really lacking in abscissic acid. Another thing that ABA does that has a growth inhibiting effect but it's super important for the survival of the plant is that ABA will signal the closure of the guard cells and the closure of the stomates, stomata, in response to wilting or any kind of drought stress that might be perceived in the roots. ABA be transmitted, for example, to the roots to the shoot before it starts to wilt. And this sets in motion the release of potassium ions from the guard cells. So the solute concentration is reduced in the guard cells and water moves out of the guard cells based on the osmotic response. So that's crucial for plants to keep their water balance intact. And ABA is the important chemical signal in this regard. So again, abscissic acid is important for dormancy, closing of stomata, and in general it's a growth inhibitor. And it's this balance of ABA and gibberellens that's really important in many cases. All right, so ethylene is a substance that was only recognized to be a plant growth regulator relatively late. And that's because it functions in the gas phase, which is a bizarre thing for a hormone to do. But this is a very important molecule for signaling the onset of, for example, leaf senescence and abscission in deciduous plants, like deciduous hardwood trees. And deciduous plants have what's called an abscission layer. It's a little layer of parenchyma at the base of the leaf. There are no fibers, scarified cells that cross through that zone. So these are just living cells that have relatively thin cell walls. And ethylene, when it's produced, what happens first is that it actually results in a programmed death of cells inside the leaf. So the leaf will undergo this process where the nutrients that are contained within that leaf are mobilized by the breakdown of substances in the leaf. And then those are moved back into the main body of the plant. And then the abscission layer changes start to occur there where ethylene will stimulate the production of enzymes that break down the cell walls of those parenchyma cells in that abscission layer, such that eventually the leaf will just fall off based on its own weight or based on wind. And then cork will form over that wound, so that prevents infection of the plant. So ethylene is a really important effect in programmed cell death that can be important in capturing back the nutrients that a plant needs from tissues that are going to be discarded. And it also has a really important effect in fruit ripening. So ethylene is crucial in fruit ripening response. So it's involved in both the enzymatic breakdown of the cell walls. It signals the production of enzymes that break down the cell walls in the fruit, which makes the fruit soft and edible in that sense. And it also results in the production of enzymes that convert starch to sugar. So it makes the fruit sweet as well. So the fruit becomes attractive to animals that disperse the seeds as a result. And this is one of the few cases in biology of a positive feedback mechanism where the production of ethylene leads to ripening. Ripening then triggers more ethylene production. So this ethylene production is increased. And as a gas, ethylene produced by one fruit can trigger the ripening of other fruit in close proximity, especially if they're in closed containers like paper bag, for example. So you've no doubt had that experience where one fruit can be important with regard to the ripening of others. And so ethylene, since it was discovered as being important in this regard, it's used commercially to allow for the picking of fruit when it's still unripe and transporting it while it's unripe so it's not damaged during transport and that it lasts. And then ethylene gas is applied after the fruit's ready to be marketed and the fruit will start ripening at that point. And you can store fruit for long periods of time for many months in an unripe state as long as you can control the ethylene production by the fruit. And so there are various filtration systems that are commercially available to remove ethylene. Carbon dioxide is also applied to remove ethylene. So this is an important commercial substance as well. Okay, so ethylene then is really important in both leaf obsession and fruit ripening among other things. So that's really all I want to say about plant hormones per se. We can just summarize them here. Of course they can have a variety of effects depending on the site of action, concentration of the hormone, for example. And this is basically what I'm saying here. Different plant parts have different sensitivities and different concentrations. So this whole notion of differential sensitivity is really important. And finally, they often act together. I've already mentioned this with regard to oxen and cytokinins working together. This hormone balance in regard to those two and then the balance in regard to gibberellins and ABA. So this makes them really difficult to study, of course. It's in many ways more difficult than studying animal hormone action. And there's a lot of activity still going on in terms of trying to understand how plant hormones operate. But it's pretty clear that hormonal balance is super important in plants. Okay, so in the last few minutes I just want to mention that plant chemistry can also be really important in terms of plant defense. And of course plants are in a real bind when it comes to defending themselves when they can't get up and move. They can't ward off an enemy or an attacker by getting away from it. So they have evolved a number of really amazing strategies. And I've just broken these into microbial attack versus attack of a macro herbivore. But in terms of, say, a bacterium or a fungus that, say, might invade the tissue through the stomata, which is an opening, a natural opening that plants have to keep open. But it does make them vulnerable. Their cuticle can ward off a lot of microbes, but the stomates are one place where they can be attacked. A particular microbe entering through the stomates can be recognized by plants. And there are proteins that are produced by plant pathogens, disease-causing agents, where we have essentially an arms race going on between the attacker and the plant in terms of the plant's ability to recognize that attacker. So much like we do in terms of disease response. And plants, if they recognize the attacker, they often have a very local and specific response to that particular agent. And they can produce a natural pesticide that will be deadly to that particular agent. And at the same time, they can undergo programmed cell death in the region of that attack. So they can wall off that part of their body that's being attacked by a microbe and at the same time that they're attacking it with a natural pesticide. So this is what we call a hypersensitive response on the part of the plant. Okay, so that's one way in which they can protect themselves from microbes. And when they're undergoing an attack like that, this is also going to result in general stimulation of plant defense genes in general, not just the specific ones, but other plant defense genes. And this can result in protection against the diversity of pathogens that can last for a number of days. This is what we call systemic acquired resistance by the plant. So we have both of these things going on in the case of a microbial attack. Okay, as far as herbivores go, so big chomping animals. For example, arthropods like insects or mites or larger vertebrate herbivores. These can be warded off by physical defenses, of course, like the cuticle, spines, things like that. Hairs on the leaf that are difficult for an insect to penetrate or glandular exudates they produce. But those glandular exudates and chemistry that's inside the leaf of the plant can also be important in warding off herbivores by producing these substances which are called secondary metabolites, which are just basically any chemical that's produced by a plant that's not involved, it's not necessary for survival and reproduction of the plant per se. There's a vast diversity of chemicals that plants produce that are not involved in primary metabolism. And when I was an undergraduate, the typical plant ecologist was unwilling to accept that these chemicals weren't just waste products. You know, plants can't easily excrete their waste, so these are just accumulated waste products. But it turns out that these chemicals often have a really important in warding off attack by particular herbivores. They're either toxic, directly toxic to the herbivore, or they're distasteful. And in general, they're harmful to their enemies. Also, in some cases, the actual attack, the saliva, for example, of the attacker is recognized by the plant and the plant can produce volatile airborne chemicals that will signal that this attacker is present. And here we can see, for example, some mites that are attacking some tomatoes, volatile chemicals produced. For example, here is a predatory mite picks up on this volatile chemical that's carried in the air and that initiates a response on the part of the mite to go to the tomato plant because that's like a ringing the food bell, means that there's a tasty item on those tomato plants and the predatory mites then come to the rescue. So this is essentially like a chemical call for help by the plant. Of course, the predatory mite doesn't really have altruistic intentions to go to rescue the plant, but it rescues the plant anyways by coming and eating its attacker. So plants have all kinds of physical defenses, chemical defenses like alkaloids, terpenes and other secondary chemicals that we're familiar with that can be highly toxic to us or can be useful in various ways. And they even can basically bring on an attacker or they can warn neighboring plants about the presence of an attacker that can also help with regard to, for example, signaling close relatives of the presence of an attacker. So plants do a lot of amazing things and I hope that this part of the course has been interesting to you and if you do have more interest in learning more about plant and fungal diversity or ecology and evolution, there's a lot of courses offered here at Cal because we have the Natural History Museums. There are a lot of faculty here that study organisms of great diversity and these are just a few of the courses that on behalf of myself and the other instructors I want to strongly encourage you to consider taking after you pass this course, which is the major prerequisite for most of these courses. So it's been a pleasure having the course and good luck with studying. Hope to see you Wednesday.