Plant Physiology Chapter 18: Blue Light Responses: Stomatal Movements and Morphogenesis PDF

Chapter Blue-Light Responses: 18 Stomatal Movements and Morphogenesis MOST OF US are familiar with the observation that house plants placed near a window have branches that grow toward the incoming light. This response, called phototropism,...

Chapter Blue-Light Responses: 18 Stomatal Movements and Morphogenesis MOST OF US are familiar with the observation that house plants placed near a window have branches that grow toward the incoming light. This response, called phototropism, is an example of how plants alter their growth patterns in response to the direction of incident radiation. This response to light is intrinsically different from light trapping by photo- synthesis. In photosynthesis, plants harness light and convert it into chemical energy (see Chapters 7 and 8). In contrast, phototropism is an example of the use of light as an environmental signal. There are two major families of plant responses to light signals: the phytochrome responses, which were covered in Chapter 17, and the blue-light responses. Some blue-light responses were introduced in Chapter 9—for exam- ple, chloroplast movement within cells in response to incident photon fluxes, and sun tracking by leaves. As with the family of the phy- tochrome responses, there are numerous plant responses to blue light. Besides phototropism, they include inhibition of hypocotyl elongation, stimulation of chlorophyll and carotenoid synthesis, activation of gene expression, stomatal movements, phototaxis (the movement of motile unicellular organisms such as algae and bacteria toward or away from light), enhancement of respiration, and anion uptake in algae (Senger 1984). Blue-light responses have been reported in higher plants, algae, ferns, fungi, and prokaryotes. Some responses, such as electrical events at the plasma membrane, can be detected within seconds of irradiation by blue light. More complex metabolic or morphogenetic responses, such as blue light–stimulated pig- ment biosynthesis in the fungus Neurospora or branching in the alga Vaucheria, might require minutes, hours, or even days (Horwitz 1994). Readers may be puzzled by the different approaches to naming phy- tochrome and blue-light responses. The former are identified by a spe- cific photoreceptor (phytochrome), the latter by the blue-light region of the visible spectrum. In the case of phytochrome, several of its spectro- scopic and biochemical properties, particularly its red/far-red reversibil- 404 Chapter 18 ity, made possible its early identification, and hundreds of to light that are mediated by photosynthesis, phytochrome, photobiological responses of plants can be clearly attrib- or other photoreceptors (Cosgrove 1994). uted to the phytochrome photoreceptor (see Chapter 17). In this chapter we will describe representative blue-light In contrast, the spectroscopy of blue-light responses is responses in plants: phototropism, inhibition of stem elon- complex. Both chlorophylls and phytochrome absorb blue gation, and stomatal movements. The stomatal responses light (400–500 nm) from the visible spectrum, and other to blue light are discussed in detail because of the impor- chromophores and some amino acids, such as tryptophan, tance of stomata in leaf gas exchange (see Chapter 9) and absorb light in the ultraviolet (250–400 nm) region. How, in plant acclimations and adaptations to their environment. then, can we then distinguish specific responses to blue We will also discuss blue-light photoreceptors and the sig- light? One important identification criterion is that in spe- nal transduction cascade that links light perception with cific blue-light responses, blue light cannot be replaced by the final expression of blue-light sensing in the organism. a red-light treatment, and there is no red/far-red reversibil- ity. Red or far-red light would be effective if photosynthe- sis or phytochrome were involved. THE PHOTOPHYSIOLOGY OF Another key distinction is that many blue-light responses BLUE-LIGHT RESPONSES of higher plants share a characteristic action spectrum. You will Blue-light signals are utilized by the plant in many recall from Chapter 7 that an action spectrum is a graph of responses, allowing the plant to sense the presence of light the magnitude of the observed light response as a function and its direction. This section describes the major mor- of wavelength (see Web Topic 7.1 for a detailed discussion phological, physiological, and biochemical changes associ- of spectroscopy and action spectra). The action spectrum ated with typical blue-light responses. of the response can be compared with the absorption spectra of candidate photoreceptors. A close correspondence Blue Light Stimulates Asymmetric Growth and between action and absorption spectra provides a strong Bending indication that the pigment under consideration is the pho- Directional growth toward (or in special circumstances toreceptor mediating the light response under study (see away from) the light, is called phototropism. It can be Figure 7.8). observed in fungi, ferns, and higher plants. Phototropism Action spectra for blue light–stimulated phototropism, is a photomorphogenetic response that is particularly dra- stomatal movements, inhibition of hypocotyl elongation, matic in dark-grown seedlings of both monocots and and other key blue-light responses share a characteristic dicots. Unilateral light is commonly used in experimental “three-finger” fine structure in the 400 to 500 nm region studies, but phototropism can also be observed when a (Figure 18.1) that is not observed in spectra for responses seedling is exposed to two unequally bright light sources (Figure 18.2), a condition that can occur in nature. As it grows through the soil, the shoot of a grass is pro- tected by a modified leaf that covers it, called a coleoptile 1.40 Blue region of spectrum (Figure 18.3; see also Figure 19.1). As discussed in detail in Curvature per photon, relative to 436 nm Chapter 19, unequal light perception in the coleoptile 1.20 results in unequal concentrations of auxin in the lighted 1.00 and shaded sides of the coleoptile, unequal growth, and bending. 0.80 Keep in mind that phototropic bending occurs only in growing organs, and that coleoptiles and shoots that have 0.60 stopped elongating will not bend when exposed to unilat- eral light. In grass seedlings growing in soil under sunlight, 0.40 coleoptiles stop growing as soon as the shoot has emerged from the soil and the first true leaf has pierced the tip of the 0.20 coleoptile. On the other hand, dark-grown, etiolated coleoptiles con- 0 tinue to elongate at high rates for several days and, 300 320 340 360 380 400 420 440 460 480 500 Wavelength (nm) depending on the species, can attain several centimeters in length. The large phototropic response of these etiolated FIGURE 18.1 Action spectrum for blue light–stimulated coleoptiles (see Figure 18.3) has made them a classic model phototropism in oat coleoptiles. An action spectrum shows for studies of phototropism (Firn 1994). the relationship between a biological response and the wavelengths of light absorbed. The “three-finger” pattern The action spectrum shown in Figure 18.1 was obtained in the 400 to 500 nm region is characteristic of specific blue- through measurement of the angles of curvature from oat light responses. (After Thimann and Curry 1960.) coleoptiles that were irradiated with light of different  Blue-Light Responses: Stomatal Movements and Morphogenesis 405 Light source wavelengths. The spectrum shows a peak at about 370 nm Direction of growth and the “three-finger” pattern in the 400 to 500 nm region Cotyledons discussed earlier. An action spectrum for phototropism in the dicot alfalfa (Medicago sativa) was found to be very sim- ilar to that of oat coleoptiles, suggesting that a common photoreceptor mediates phototropism in the two species. Phototropism in sporangiophores of the mold Phy- Unilateral light Unequal bilateral illumination comyces has been studied to identify genes involved in pho- totropic responses. The sporangiophore consists of a spo- rangium (spore-bearing spherical structure) that develops on a stalk consisting of a long, single cell. Growth in the sporangiophore is restricted to a growing zone just below the sporangium. When irradiated with unilateral blue light, the sporan- giophore bends toward the light with an action spectrum similar to that of coleoptile phototropism (Cerda-Olmedo and Lipson 1987). These studies of Phycomyces have led to the isolation of many mutants with altered phototropic Two equal lights from Two unequal lights from responses and the identification of several genes that are the side the side required for normal phototropism. FIGURE 18.2 Relationship between direction of growth and In recent years, phototropism of the stem of the small unequal incident light. Cotyledons from a young seedling dicot Arabidopsis (Figure 18.4) has attracted much attention are shown as viewed from the top. The arrows indicate the because of the ease with which advanced molecular tech- direction of phototropic curvature. The diagrams illustrate niques can be applied to Arabidopsis mutants. The genetics how the direction of growth varies with the location and and the molecular biology of phototropism in Arabidopsis the intensity of the light source, but growth is always toward light. (After Firn 1994.) are discussed later in this chapter. (A) Wild-type Blue light (B) Mutant Blue light FIGURE 18.3 Time-lapse photograph of a corn coleoptile growing toward unilateral blue light given from the right. The consecutive exposures were made 30 minutes apart. FIGURE 18.4 Phototropism in wild-type (A) and mutant (B) Note the increasing angle of curvature as the coleoptile Arabidopsis seedlings. Unilateral light was applied from the bends. (Courtesy of M. A. Quiñones.) right. (Courtesy of Dr. Eva Huala.) 406 Chapter 18 How Do Plants Sense the Direction of the Light However, action spectra for the decrease in elongation Signal? rate show strong activity in the blue region, which cannot Light gradients between lighted and shaded sides have been be explained by the absorption properties of phytochrome measured in coleoptiles and in hypocotyls from dicot (see Figure 17.9). In fact, the 400 to 500 nm blue region of seedlings irradiated with unilateral blue light. When a the action spectrum for the inhibition of stem elongation coleoptile is illuminated with 450 nm blue light, the ratio closely resembles that of phototropism (compare the action between the light that is incident to the surface of the illu- spectra in Figures 17.10 and 18.1). minated side and the light that reaches the shaded side is There are several ways to experimentally separate a 4:1 at the tip and the midregion of the coleoptile, and 8:1 at reduction in elongation rates mediated by phytochrome the base (Figure 18.5). from a reduction mediated by a specific blue-light response. On the other hand, there is a lens effect in the sporangio- If lettuce seedlings are given low fluence rates of blue light phore of the mold Phycomyces irradiated with unilateral under a strong background of yellow light, their hypocotyl blue light, and as a result, the light measured at the distal elongation rate is reduced by more than 50%. The back- cell surface of the sporangiophore is about twice the ground yellow light establishes a well-defined Pr:Pfr ratio amount of light that is incident at the surface of the illumi- (see Chapter 17). In such conditions, the low fluence rates nated side. Light gradients and lens effects could play a of blue light added are too small to significantly change this role in how the bending organ senses the direction of the ratio, ruling out a phytochrome effect on the reduction in unilateral light (Vogelmann 1994). elongation rate observed upon the addition of blue light. Blue light– and phytochrome-mediated hypocotyl Blue Light Rapidly Inhibits Stem Elongation responses can also be distinguished by the swiftness of the The stems of seedlings growing in the dark elongate very response. Whereas phytochrome-mediated changes in rapidly, and the inhibition of stem elongation by light is a elongation rates can be detected within 8 to 90 minutes, key morphogenetic response of the seedling emerging depending on the species, blue-light responses are rapid, from the soil surface (see Chapter 17). The conversion of and can be measured within 15 to 30 s (Figure 18.6). Inter- Pr to Pfr (the red- and far red–absorbing forms of phy- actions between phytochrome and the blue light–depen- tochrome, respectively) in etiolated seedlings causes a dent sensory transduction cascade in the regulation of elon- phytochrome-dependent, sharp decrease in elongation gation rates will be described later in the chapter. rates (see Figure 17.1). Another fast response elicited by blue light is a depo- larization of the membrane of hypocotyl cells that precedes the inhibition of growth rate (see Figure 18.6). The membrane depolarization is caused by the activation of anion channels (see Chapter 6), which facilitates the efflux of anions such as chlo- Probe Probe ride. Use of an anion channel blocker prevents the blue light–dependent membrane depolarization 1.2 Blue Blue light light and decreases the inhibitory effect of blue light on Light (relative units) 0.8 hypocotyl elongation (Parks et al. 1998). 0.4 Blue Light Regulates Gene Expression Blue light also regulates the expression of genes 0 involved in several important morphogenetic processes. Some of these light-activated genes have been studied in detail—for example, the genes that code for the enzyme chalcone synthase, which cat- 0 1.0 0 1.0 2.0 alyzes the first committed step in flavonoid biosyn- Distance (mm) thesis, for the small subunit of rubisco, and for the proteins that bind chlorophylls a and b (see Chap- FIGURE 18.5 Distribution of transmitted, 450 nm blue light in an etiolated corn coleoptile. The diagram in the upper right of each ters 13, 8, and 7, respectively). Most of the studies frame shows the area of the coleoptile being measured by a fiber- on light-activated genes show sensitivity to both optic probe. A cross section of the tissue appears at the bottom of blue and red light, as well as red/far-red reversibil- each frame. The trace above it shows the amount of light sensed by ity, implicating both phytochrome and specific blue- the probe at each point. A sensing mechanism that depended on light responses. light gradients would sense the difference in the amount of light between the lighted and shaded sides of the coleoptile, and this A recent study reported that SIG5, one of six SIG information would be transduced into an unequal auxin concen- nuclear genes in Arabidopsis that play a regulatory tration and bending. (After Vogelmann and Haupt 1985.) role in the transcription of the chloroplast gene  Blue-Light Responses: Stomatal Movements and Morphogenesis 407 (A) the onset of illumination, GSA mRNA levels are 26-fold 2.5 Blue light on higher than they are in the dark (Figure 18.7). These blue light–mediated mRNA increases precede increases in Growth rate (mm h–1) chlorophyll content, indicating that chlorophyll biosyn- 2.0 thesis is being regulated by activation of the GSA gene. Blue Light Stimulates Stomatal Opening 1.5 We now turn our attention to the stomatal response to blue light. Stomata have a major regulatory role in gas exchange in leaves (see Chapter 9), and they can often affect yields of agricultural crops (see Chapter 25). Several characteristics 1.0 of blue light–dependent stomatal movements make guard cells a valuable experimental system for the study of blue- (B) 0 1 2 3 4 light responses: –60 The stomatal response to blue light is rapid and Membrane potential difference (mV) reversible, and it is localized in a single cell type, the –80 guard cell. The stomatal response to blue light regulates stom- –100 atal movements throughout the life of the plant. This is unlike phototropism or hypocotyl elongation, which are functionally important at early stages of –120 development. The signal transduction cascade that links the percep- –140 tion of blue light with the opening of stomata is understood in considerable detail. –160 0 1 2 3 4 In the following sections we will discuss two central Time (min) aspects of the stomatal response to light, the osmoregula- tory mechanisms that drive stomatal movements, and the FIGURE 18.6 Blue light–induced (A) changes in elongation role of a blue light–activated H+-ATPase in ion uptake by rates of etiolated cucumber seedlings and (B) transient membrane depolarization of hypocotyl cells. As the mem- guard cells. brane depolarization (measured with intracellular elec- trodes) reaches its maximum, growth rate (measured with position transducers) declines sharply. Comparison of the two curves shows that the membrane starts to depolarize before the growth rate begins to decline, suggesting a cause–effect relation between the two phenomena. (After Relative abundance of GSA mRNA Spalding and Cosgrove 1989.) psbD, which encodes the D2 subunit of the PSII reaction center (see Chapter 7), is specifically activated by blue light (Tsunoyama et al. 2002). In contrast, the other five SIG genes are activated by both blue and red light. Blue Another well-documented instance of gene expression light that is mediated solely by a blue light–sensing system on involves the GSA gene in the photosynthetic unicellular alga Chlamydomonas reinhardtii (Matters and Beale 1995). This gene encodes the enzyme glutamate-1-semialdehyde –2 0 2 4 6 8 10 12 aminotransferase (GSA), a key enzyme in the chlorophyll Time of blue-light treatment (h) biosynthesis pathway (see Chapter 7). The absence of phy- tochrome in C. reinhardtii simplifies the analysis of blue- FIGURE 18.7 Time course of blue light–dependent gene expression in Chlamydomonas reinhardtii. The GSA gene light responses in this experimental system. encodes the enzyme glutamate-1-semialdehyde amino- In synchronized cultures of C. reinhardtii, levels of GSA transferase, which regulates an early step in chlorophyll mRNA are strictly regulated by blue light, and 2 hours after biosynthesis. (After Matters and Beale 1995.) 408 Chapter 18 (A) (B) broad bean (Vicia faba), stomatal movements closely track incident solar radiation at the leaf surface (Figure 18.9). Chloroplast Pore Early studies of the stomatal response to light showed that DCMU (dichlorophenyl- dimethylurea), an inhibitor of photosynthetic electron transport (see Figure 7.31), causes a partial inhibition of light-stimulated stomatal opening. These results indicated that photo- synthesis in the guard cell chloroplast plays a role in light-dependent stomatal opening, but the observation that the inhibition was only partial pointed to a nonphotosynthetic compo- nent of the stomatal response to light. Detailed studies of the light response of stomata have shown that light activates two distinct Guard cells responses of guard cells: photosynthesis in the guard cell chloroplast (see Web Essay 18.1), 20 µm and a specific blue-light response. FIGURE 18.8 Light-stimulated stomatal opening in detached epidermis The specific stomatal response to blue light of Vicia faba. Open, light-treated stoma (A), is shown in the dark- cannot be resolved properly under blue-light treated, closed state in (B). Stomatal opening is quantified by micro- illumination because blue light simultaneously scopic measurement of the width of the stomatal pore. (Courtesy of stimulates both the specific blue-light response E. Raveh.) and guard cell photosynthesis (for the photo- synthetic response to blue light, see the action Light is the dominant environmental signal controlling spectrum for photosynthesis in Figure 7.8). A clear-cut sep- stomatal movements in leaves of well-watered plants aration of the responses of the two light responses can be growing in natural environments. Stomata open as light obtained in dual-beam experiments. High fluence rates of levels reaching the leaf surface increase, and close as they red light are used to saturate the photosynthetic response, decrease (Figure 18.8). In greenhouse-grown leaves of and low photon fluxes of blue light are added after the response to the saturating red light has been completed (Figure 18.10). The addition of blue light causes substantial further stomatal opening that cannot be explained as a fur- (A) 1250 ther stimulation of guard cell photosynthesis because pho- Photosynthetically active tosynthesis is saturated by the background red light. radiation (400–700 nm) 1000 An action spectrum for the stomatal response to blue 750 light under background red illumination shows the three- finger pattern discussed earlier (Figure 18.11). This action (µmol m–2 s–1) 500 spectrum, typical of blue-light responses and distinctly dif- ferent from the action spectrum for photosynthesis, further 250 indicates that, in addition to photosynthesis, guard cells respond specifically to blue light. 0 When guard cells are treated with cellulolytic enzymes (B) 14 that digest the cell walls, guard cell protoplasts are released. 12 Guard cell protoplasts swell when illuminated with blue Stomatal aperture light (Figure 18.12), indicating that blue light is sensed (pore width, µm) 10 within the guard cells proper. The swelling of guard cell 8 6 FIGURE 18.9 Stomatal opening tracks photosynthetic active radiation at 4 the leaf surface. Stomatal opening in the lower surface of leaves of Vicia faba grown in a greenhouse, measured as the width of the stomatal pore 2 (A), closely follows the levels of photosynthetically active radiation 0 (400–700 nm) incident to the leaf (B), indicating that the response to light 5:00 9:00 13:00 17:00 21:00 was the dominant response regulating stomatal opening. (After Srivastava Time of day and Zeiger 1995a.)  Blue-Light Responses: Stomatal Movements and Morphogenesis 409 (A) 12 Blue Stomatal aperture (µm) 10 light 8 Blue light 6 4 2 Red light Undigested stomatal pore 0 1 2 3 4 Time (h) Protoplasts in dark Protoplasts swell in FIGURE 18.10 The response of stomata to blue light under a blue light red-light background. Stomata from detached epidermis of Commelina communis (common dayflower) were treated (B) with saturating photon fluxes of red light (red trace). In a Guard cell protoplast volume (µm3 × 10–2) 55 parallel treatment, stomata illuminated with red light were Red light on also illuminated with blue light, as indicated by the arrow Blue light on 50 (blue trace). The increase in stomatal opening above the level reached in the presence of saturating red light indi- Control cates that a different photoreceptor system, stimulated by 45 blue light, is mediating the additional increases in opening. 500 µM (From Schwartz and Zeiger 1984.) 40 Vanadate 35 protoplasts also illustrates how intact guard cells function. 30 The light-stimulated uptake of ions and the accumulation of organic solutes decrease the cell’s osmotic potential (increase the osmotic pressure). Water flows in as a result, 0 20 40 60 leading to an increase in turgor that in guard cells with Time (min) intact walls is mechanically transduced into an increase in stomatal apertures (see Chapter 4). In the absence of a cell FIGURE 18.12 Blue light–stimulated swelling of guard cell wall, the blue light–mediated increase in osmotic pressure protoplasts. (A) In the absence of a rigid cell wall, guard causes the guard cell protoplast to swell. cell protoplasts of onion (Allium cepa) swell. (B) Blue light stimulates the swelling of guard cell protoplasts of broad bean (Vicia faba), and vanadate, an inhibitor of the H+- ATPase, inhibits this swelling. Blue light stimulates ion and water uptake in the guard cell protoplasts, which in the intact guard cells provides a mechanical force that drives increases in stomatal apertures. (A from Zeiger and Hepler 1977; B after Amodeo et al. 1992.) Relative effectiveness Blue Light Activates a Proton Pump at the Guard Cell Plasma Membrane When guard cell protoplasts from broad bean (Vicia faba) are irradiated with blue light under background red-light illumination, the pH of the suspension medium becomes 350 400 450 500 more acidic (Figure 18.13). This blue light–induced acidifi- Wavelength (nm) cation is blocked by inhibitors that dissipate pH gradients, FIGURE 18.11 The action spectrum for blue light–stimu- such as CCCP (discussed shortly), and by inhibitors of the lated stomatal opening (under a red-light background). proton-pumping H+-ATPase, such as vanadate (see Figure (After Karlsson 1986.) 18.12C; see also Chapter 6). 410 Chapter 18 More (A) alkaline CCCP proton ionophore Electric current Baseline under Blue-light pH of suspension medium saturating red pulse light Blue photon fluxes (µmol m–2 s–1): 5 Fusicoccin activates H+-ATPase 2 pA 10 50 500 1 min (B) More 0 10 20 30 40 50 60 acidic Time (min) Electric current FIGURE 18.13 Acidification of a suspension medium of guard cell protoplasts of Vicia faba stimulated by a 30 s pulse of blue light. The acidification results from the stimu- lation of an H+-ATPase at the plasma membrane by blue light, and it is associated with protoplast swelling (see Figure 18.12). (After Shimazaki et al. 1986.) Blue-light pulse 2 pA 30 s This indicates that the acidification results from the activa- tion by blue light of a proton-pumping ATPase in the guard cell FIGURE 18.14 Activation of the H+-ATPase at the plasma plasma membrane that extrudes protons into the protoplast membrane of guard cell protoplasts by fusiccocin and blue light can be measured as electric current in patch clamp suspension medium and lowers its pH. In the intact leaf, experiments. (A) Outward electric current (measured in this blue-light stimulation of proton pumping lowers the picoamps, pA) at the plasma membrane of a guard cell pro- pH of the apoplastic space surrounding the guard cells. toplast stimulated by the fungal toxin fusicoccin, an activa- The plasma membrane ATPase from guard cells has been tor of the H+-ATPase. The current is abolished by the pro- isolated and extensively characterized (Kinoshita et al. ton ionophore CCCP (carbonyl cyanide m-chlorophenylhy- drazone). (B) Outward electric current at the plasma mem- 2001). brane of a guard cell protoplast stimulated by a blue-light The activation of electrogenic pumps such as the proton- pulse. These results indicate that blue light stimulates the pumping ATPase can be measured in patch-clamping H+-ATPase. (A after Serrano et al. 1988; B after Assmann et experiments as an outward electric current at the plasma al. 1985.) membrane (see Web Topic 6.2 for a description of patch clamping). A patch clamp recording of a guard cell proto- plast treated with the fungal toxin fusicoccin, a well-char- acterized activator of plasma membrane ATPases, is shown The close relationship among the number of incident in Figure 18.14A. Exposure to fusicoccin stimulates an out- blue-light photons, proton pumping at the guard cell ward electric current, which is abolished by the proton plasma membrane, and stomatal opening further suggests ionophore carbonyl cyanide m-chlorophenylhydrazone that the blue-light response of stomata might function as a (CCCP). This proton ionophore makes the plasma mem- sensor of photon fluxes reaching the guard cell. brane highly permeable to protons, thus precluding the for- Pulses of blue light given under a saturating red-light mation of a proton gradient across the membrane and abol- background also stimulate an outward electric current from ishing net proton efflux. guard cell protoplasts (see Figure 18.14B). The acidification The relationship between proton pumping at the guard measurements shown in Figure 18.13 indicate that the out- cell plasma membrane and stomatal opening is evident ward electric current measured in patch clamp experiments from the observation that fusicoccin stimulates both pro- is carried by protons. ton extrusion from guard cell protoplasts and stomatal opening, and that CCCP inhibits the fusiccocin-stimulated Blue-Light Responses Have Characteristic opening. The increase in proton-pumping rates as a func- Kinetics and Lag Times tion of fluence rates of blue light (see Figure 18.13) indicates Some of the characteristics of the responses to blue-light that the increasing rates of blue photons in the solar radia- pulses underscore some important properties of blue-light tion reaching the leaf cause a larger stomatal opening. responses: the persistence of the response after the light sig-  Blue-Light Responses: Stomatal Movements and Morphogenesis 411 nal has been switched off, and a significant lag time sepa- The Cl– ion is taken up into the guard cells during stom- rating the onset of the light signal and the beginning of the atal opening and extruded during stomatal closing. Malate, response. on the other hand, is synthesized in the guard cell cytosol, In contrast to typical photosynthetic responses, which in a metabolic pathway that uses carbon skeletons gener- are activated very quickly after a “light on” signal, and ated by starch hydrolysis (see Figure 18.15B). The malate cease when the light goes off (see, for instance, Figure 7.13), content of guard cells decreases during stomatal closing, blue-light responses proceed at maximal rates for several but it remains to be established whether malate is catabo- minutes after application of the pulse (see Figure 18.14B). lized in mitochondrial respiration or is extruded into the This property can be explained by a physiologically inac- apoplast. tive form of the blue-light photoreceptor that is converted Potassium and chloride are taken up into guard cells via to an active form by blue light, with the active form revert- secondary transport mechanisms driven by the gradient of ing slowly to the physiologically inactive form in the electrochemical potential for H+, ∆mH+, generated by the absence of blue light (Iino et al. 1985). The rate of the proton pump (see Chapter 6) discussed earlier in the chap- response to a blue-light pulse would thus depend on the ter. Proton extrusion makes the electric-potential difference time course of the reversion of the active form to the inac- across the guard cell plasma membrane more negative; tive one. light-dependent hyperpolarizations as high as 50 mV have Another property of the response to blue-light pulses is been measured. In addition, proton pumping generates a a lag time, which lasts about 25 s in both the acidification pH gradient of about 0.5 to 1 pH unit. response and the outward electric currents stimulated by The electrical component of the proton gradient pro- blue light (see Figures 18.13 and 18.14). This amount of vides a driving force for the passive uptake of potassium time is probably required for the signal transduction cas- ions via voltage-regulated potassium channels (see Chap- cade to proceed from the photoreceptor site to the proton- ter 6) (Schroeder et al. 2001). Chloride is thought to be pumping ATPase and for the proton gradient to form. Sim- taken up through anion channels. Thus, blue light–depen- ilar lag times have been measured for blue light–dependent dent stimulation of proton pumping plays a key role in inhibition of hypocotyl elongation, which was discussed guard cell osmoregulation during light-dependent stom- earlier. atal movements Guard cell chloroplasts (see Figure 18.8) contain large Blue Light Regulates Osmotic Relations starch grains, and their starch content decreases during of Guard Cells stomatal opening and increases during closing. Starch, an Blue light modulates guard cell osmoregulation via its acti- insoluble, high-molecular-weight polymer of glucose, does vation of proton pumping (described earlier) and via the not contribute to the cell’s osmotic potential, but the stimulation of the synthesis of organic solutes. Before dis- hydrolysis of starch into soluble sugars causes a decrease cussing these blue-light responses, let us briefly describe in the osmotic potential (or increase in osmotic pressure) of the major osmotically active solutes in guard cells. guard cells. In the reverse process, starch synthesis The botanist Hugo von Mohl proposed in 1856 that tur- decreases the sugar concentration, resulting in an increase gor changes in guard cells provide the mechanical force for of the cell’s osmotic potential, which the starch–sugar changes in stomatal apertures. The plant physiologist F. E. hypothesis predicted to be associated with stomatal clos- Lloyd hypothesized in 1908 that guard cell turgor is regu- ing. lated by osmotic changes resulting from starch–sugar inter- With the discovery of the major role of potassium and conversions, a concept that led to a starch–sugar hypoth- its counterion in guard cell osmoregulation, the sugar– esis of stomatal movements. The discovery of the changes starch hypothesis was no longer considered important in potassium concentrations in guard cells in the 1960s led (Outlaw 1983). Recent studies, however, described in the to the modern theory of guard cell osmoregulation by next section, have characterized a major osmoregulatory potassium and its counterions. phase of guard cells in which sucrose is the dominant Potassium concentration in guard cells increases sever- osmotically active solute. alfold when stomata open, from 100 mM in the closed state to 400 to 800 mM in the open state, depending on the plant Sucrose Is an Osmotically Active Solute species and the experimental conditions. These large con- in Guard Cells centration changes in the positively charged potassium Studies of daily courses of stomatal movements in intact ions are electrically balanced by the anions Cl– and leaves have shown that the potassium content in guard malate2– (Figure 18.15A). In species of the genus Allium, cells increases in parallel with early-morning opening, but such as onion (Allium cepa), K+ ions are balanced solely by it decreases in the early afternoon under conditions in Cl–. In most species, however, potassium fluxes are bal- which apertures continue to increase. The sucrose content anced by varying amounts of Cl– and the organic anion of guard cells increases slowly in the morning, but upon malate2– (Talbott et al. 1996). potassium efflux, sucrose becomes the dominant osmoti- 412 Chapter 18 (A) CYTOPLASM CHLOROPLAST Ribulose-1,5- Fructose-6-phosphate Glucose-6-phosphate Starch bisphosphate Fructose-1,6-bisphosphate CO2 Calvin Glucose Maltose cycle Dihydroxyacetone 3-phosphate 3 phosphoglycerate Cl– Cl– CO2 H+ H+ Glucose-1-phosphate Dihydroxyacetone 3-phosphate Phosphoenol- Malate pyruvate K+ K+ VACUOLE ? Sucrose Sucrose Sucrose Malate Cl– K+ (B) CYTOPLASM CHLOROPLAST Ribulose-1,5- Fructose-6-phosphate Glucose-6-phosphate Starch bisphosphate Fructose-1,6-bisphosphate CO2 Calvin Glucose Maltose cycle Dihydroxyacetone 3-phosphate 3 phosphoglycerate Cl– Cl– CO2 H+ H+ Glucose-1-phosphate Dihydroxyacetone 3-phosphate Phosphoenol- Malate pyruvate K+ K+ VACUOLE ? Sucrose Sucrose Sucrose Malate Cl– K+ (C) CYTOPLASM CHLOROPLAST Ribulose-1,5- Fructose-6-phosphate Glucose-6-phosphate Starch bisphosphate Fructose-1,6-bisphosphate CO2 Calvin Glucose Maltose cycle Dihydroxyacetone 3-phosphate 3 phosphoglycerate Cl– Cl– CO2 H+ H+ Glucose-1-phosphate Dihydroxyacetone 3-phosphate Phosphoenol- Malate pyruvate K+ K+ VACUOLE ? Sucrose Sucrose Sucrose Malate Cl– K+ ▲ Blue-Light Responses: Stomatal Movements and Morphogenesis 413 FIGURE 18.15 Three distinct osmoregulatory pathways in 4. The uptake of apoplastic sucrose generated by meso- guard cells. The dark arrows identify the major metabolic phyll photosynthesis steps of each pathway that lead to the accumulation of osmotically active solutes in the guard cells. (A) Potassium and its counterions. Potassium and chloride are taken up in Depending on environmental conditions, one or several secondary transport processes driven by a proton gradient; pathways may be activated. For instance, red light–stim- malate is formed from the hydrolysis of starch. (B) ulated stomatal opening in detached epidermis depends Accumulation of sucrose from starch hydrolysis. (C) solely on sucrose generated by guard cell photosynthesis, Accumulation of sucrose from photosynthetic carbon fixa- tion. The possible uptake of apoplastic sucrose is also indi- with no detectable K+ uptake. The other osmoregulatory cated. (From Talbott and Zeiger 1998.) pathways can be selectively activated under different experimental conditions (see Web Topic 18.1). Current studies are beginning to unravel the mysteries of guard cell cally active solute, and stomatal closing at the end of the osmoregulation in the intact leaf (Dietrich et al. 2001). day parallels a decrease in the sucrose content of guard cells (Figure 18.16) (Talbott and Zeiger 1998). These osmoregulatory features indicate that stomatal BLUE-LIGHT PHOTORECEPTORS opening is associated primarily with K+ uptake, and clos- Experiments carried out by Charles Darwin and his son ing is associated with a decrease in sucrose content (see Francis in the nineteenth century determined that the site Figure 18.16). The need for distinct potassium- and sucrose- of photoreception in blue light–stimulated phototropism is dominated osmoregulatory phases is unclear, but it might in the coleoptile tip. Early hypotheses about blue-light pho- underlie regulatory aspects of stomatal function. Potassium toreceptors focused on carotenoids and flavins (for a his- might be the solute of choice for the consistent daily open- torical account of early research on blue-light photorecep- ing that occurs at sunrise. The sucrose phase might be asso- tors, see Web Topic 18.2). Despite active research efforts, ciated with the coordination of stomatal movements in the no significant advances toward the identification of blue- epidermis with rates of photosynthesis in the mesophyll. light photoreceptors were made until the early 1990s. In the Where do osmotically active solutes originate? Four dis- case of phototropism and the inhibition of stem elongation, tinct metabolic pathways that can supply osmotically progress resulted from the identification of mutants for key active solutes to guard cells have been characterized (see blue-light responses, and the subsequent isolation of the Figure 18.15): relevant gene. 1. The uptake of K+ and Cl– coupled to the biosynthesis Cloning of the gene led to the identification and char- of malate2– acterization of the protein encoded by the gene. In the case of stomatal guard cells, the carotenoid zeaxanthin has been 2. The production of sucrose from starch hydrolysis postulated to be the chromophore of a blue-light photore- 3. The production of sucrose by photosynthetic carbon ceptor, whereas the identity of the apoprotein remains to fixation in the guard cell chloroplast be established. For a detailed discussion of the basic dif- ferences between carotenoid and flavin photoreceptors, see Web Topic 18.3. In the following section we will describe Sucrose (pmol/guard cell pair) 25 2.25 the three photoreceptors associated with blue-light K+ stain (percent area) Stomatal aperture 55 responses: cryptochromes, phototropins, and zeaxanthin. Stomatal aperture (µm) 20 45 1.75 Cryptochromes Are Involved in the Inhibition of 35 Stem Elongation 15 Sucrose 1.25 K+ The hy4 mutant of Arabidopsis lacks the blue light–stimulated 25 inhibition of hypocotyl elongation described earlier in the 10 0.75 chapter. As a result of this genetic defect, hy4 plants show an 15 elongated hypocotyl when irradiated with blue light. Isola- 5 5 0.25 tion of the HY4 gene showed that it encodes a 75 kDa protein 7:00 9:00 11:00 13:00 15:00 17:00 19:00 21:00 23:00 with significant sequence homology to microbial DNA pho- tolyase, a blue light–activated enzyme that repairs pyrimi- Time of day dine dimers in DNA formed as a result of exposure to ultra- FIGURE 18.16 Daily course of changes in stomatal aperture, violet radiation (Ahmad and Cashmore 1993). In view of this and in potassium and sucrose content, of guard cells from sequence similarity, the hy4 protein, later renamed cryp- intact leaves of broad bean (Vicia faba). These results indi- tochrome 1 (cry1), was proposed to be a blue-light photore- cate that the changes in osmotic potential required for stomatal opening in the morning are mediated by potas- ceptor mediating the inhibition of stem elongation. sium and its counterions, whereas the afternoon changes Photolyases are pigment proteins that contain a flavin are mediated by sucrose. (After Talbott and Zeiger 1998.) adenine dinucleotide (FAD; see Figure 11.2B) and a pterin. 414 Chapter 18 (A) (B) response. In addition, CRY1 has been shown to be involved in the setting of the circadian clock in Arabidopsis (see Chap- Anthocyanin accumulation 0.8 Hypocotyl length (cm) ter 17), and both CRY1 and CRY2 have been shown to play 0.6 1.5 a role in the induction of flowering (see Chapter 24). Cryp- absorbance change tochrome homologs have been found to regulate the circa- 0.4 1.0 dian clock in Drosophila, mouse, and humans. 0.2 0.5 Phototropins Are Involved in Phototropism and Chloroplast Movements 0.0 Some recently isolated Arabidopsis mutants impaired in CRY1 WT cry1 CRY1 WT cry1 OE OE blue light–dependent phototropism of the hypocotyl have provided valuable information about cellular events pre- FIGURE 18.17 Blue light stimulates the accumulation of anthocyanin (A) and the inhibition of stem elongation (B) in ceding bending. One of these mutants, the nph1 (nonpho- transgenic and mutant seedlings of Arabidopsis. These bar totropic hypocotyl) mutant has been found to be genetically graphs show a transgenic phenotype overexpressing the independent of the hy4 (cry1) mutant discussed earlier: The gene that encodes CRY1 (CRY1 OE), the wild type (WT), nph1 mutant lacks a phototropic response in the hypocotyl and cry1 mutants. The enhanced blue-light response of the but has normal blue light–stimulated inhibition of transgenic plant overexpressing the gene that encodes CRY1 demonstrates the important role of this gene product hypocotyl elongation, while hy4 has the converse pheno- in stimulating anthocyanin biosynthesis and inhibiting type. Recently the nph1 gene was renamed phot1, and the stem elongation. (After Ahmad et al. 1998.) protein it encodes was named phototropin (Briggs and Christie 2002). The C-terminal half of phototropin is a serine/threonine Pterins are light-absorbing, pteridine derivatives that often kinase. The N-terminal half contains two repeated function as pigments in insects, fishes, and birds (see Chap- domains, of about 100 amino acids each, that have ter 12 for pterin structure). When expressed in Escherichia coli, sequence similarities to other proteins involved in signal- the cry1 protein binds FAD and a pterin, but it lacks ing in bacteria and mammals. Proteins with sequence sim- detectable photolyase activity. No information is available ilarity to the N terminus of phototropin bind flavin cofac- on the chromophore(s) bound to cry1 in vivo, or on the tors. These proteins are oxygen sensors in Escherichia coli nature of the photochemical reactions involving cry1, that and Azotobacter, and voltage sensors in potassium channels would start the postulated sensory transduction cascade of Drosophila and vertebrates. mediating the several blue-light responses mediated by cry1. When expressed in insect cells, the N-terminal half of The most important evidence for a role of cry1 in blue phototropin binds flavin mononucleotide (FMN) (see Fig- light–mediated inhibition of stem elongation comes from ure 11.2B and Web Essay 18.2) and shows a blue overexpression studies. Overexpression of the CRY1 pro- light–dependent autophosphorylation reaction. This reac- tein in transgenic tobacco or Arabidopsis plants results in a tion resembles the blue light–dependent phosphorylation stronger blue light–stimulated inhibition of hypocotyl of a 120 kDa membrane protein found in growing regions elongation than in the wild type, as well as increased of etiolated seedlings. production of anthocyanin, another blue-light response The Arabidopsis genome contains a second gene, phot2, (Figure 18.17). Thus, overexpression of CRY1 caused an which is related to phot1. The phot1 mutant lacks hypocotyl enhanced sensitivity to blue light in transgenic plants. phototropism in response to low-intensity blue light (0.01–1 Other blue-light responses, such as phototropism and blue µmol mol–2 s–1) but retains a phototropic response at higher light–dependent stomatal movements, appear to be nor- intensities (1–10 µmol m–2 s–1). The phot2 mutant has a nor- mal in the cry1 mutant phenotype. mal phototropic response, but the phot1/phot2 double A second gene product homologous to CRY1, named mutant is severely impaired at both low and high intensi- CRY2, has been isolated from Arabidopsis (Lin 2000). Both ties. These data indicate that both phot1 and phot2 are CRY1 and CRY2 appear ubiquitous throughout the plant involved in the phototropic response, with phot2 function- kingdom. A major difference between them is that CRY2 is ing at high light fluence rates. rapidly degraded in the light, whereas CRY1 is stable in light-grown seedlings. Blue light–activated chloroplast movement. Leaves Transgenic plants overexpressing the gene that encodes show an adaptive feature that can alter the intracellular dis- CRY2 show a small enhancement of the inhibition of tribution of their chloroplasts in order to control light hypocotyl elongation, indicating that unlike CRY1, CRY2 absorption and prevent photodamage (see Figure 9.5). The does not play a primary role in inhibiting stem elongation. action spectrum for chloroplast movement shows the On the other hand, the transgenic plants overexpressing the “three finger” fine structure typical of blue-light responses. gene that encodes CRY2 show a large increase in blue When incident radiation is weak, chloroplasts gather at the light–stimulated cotyledon expansion, yet another blue-light upper and lower surfaces of the mesophyll cells (the “accu-  Blue-Light Responses: Stomatal Movements and Morphogenesis 415 mulation” response; see Figure 9.5B), thus maximizing (A) 250 1250 Zeaxanthin (mmol mol–1 Chl a+b) light absorption. radiation (µmol m–2 s–1) Photosynthetically active Under strong light, the chloroplasts move to the cell sur- 200 1000 faces that are parallel to the incident light (the “avoidance” response; see Figure 9.5C), thus minimizing light absorp- 750 150 tion. Recent studies have shown that mesophyll cells of the Guard phot1 mutant have a normal avoidance response and a rudi- cells 100 500 mentary accumulation response. Cells from the phot2 mutant show a normal accumulation response but lack the 250 50 Mesophyll avoidance response. Cells from the phot1/phot2 double cells mutant lack both the avoidance and accumulation 0 responses (Sakai et al. 2001). These results indicate that phot2 6:00 9:00 12:00 15:00 18:00 21:00 plays a key role in the avoidance response, and that both phot1 and phot2 contribute to the accumulation response. (B) 14 The Carotenoid Zeaxanthin Mediates Blue-Light 12 Stomatal aperture (mm) Photoreception in Guard Cells 10 The carotenoid zeaxanthin has been implicated as a pho- toreceptor in blue light–stimulated stomatal opening. Recall 8 from Chapters 7 and 9 that zeaxanthin is one of the three 6 components of the xanthophyll cycle of chloroplasts, which protects photosynthetic pigments from excess excitation 4 energy. In guard cells, however, the changes in zeaxanthin 2 content as a function of incident radiation are distinctly dif- ferent from the changes in mesophyll cells (Figure 18.18). 0 6:00 9:00 12:00 15:00 18:00 21:00 In sun plants such as Vicia faba, zeaxanthin accumula- Time of day tion in the mesophyll begins at about 200 µmol m–2 s–1, and there is no detectable zeaxanthin in the early morning or FIGURE 18.18 The zeaxanthin content of guard cells closely tracks photosynthetic active radiation and stomatal aper- late afternoon. In contrast, the zeaxanthin content in guard tures. (A) Daily course of photosynthetic active radiation cells closely follows incident solar radiation at the leaf sur- reaching the leaf surface, and of zeaxanthin content of face throughout the day, and it is nearly linearly propor- guard cells and mesophyll cells of Vicia faba leaves grown in tional to incident photon fluxes in the early morning and a greenhouse. The white areas within the graph highlight late afternoon. Several key characteristics of the guard cell the contrasting sensitivity of the xanthophyll cycle in meso- phyll and guard cell chloroplasts under the low irradiances chloroplast strongly indicate that the primary function of prevailing early and late in the day. (B) Stomatal apertures the guard cell chloroplast is sensory transduction and not in the same leaves used to measure guard cell zeaxanthin carbon fixation (Zeiger et al. 2002). content. (After Srivastava and Zeiger 1995a.) Compelling evidence indicates that zeaxanthin is a blue- light photoreceptor in guard cells: The absorption spectrum of zeaxanthin (Figure 18.19) closely matches the action spectrum for blue light–stimulated stomatal opening (see Figure 18.11). In daily courses of stomatal opening in intact leaves 0.25 grown in a greenhouse, incident radiation, zeaxan- thin content of guard cells, and stomatal apertures 0.2 are closely related (see Figure 18.18). Absorbance 0.15 The blue-light sensitivity of guard cells increases as a function of their zeaxanthin concentration. 0.1 Experimentally, zeaxanthin concentration in guard cells can be varied with increasing fluence rates of 0.05 red light. When guard cells from epidermal peels illuminated with increasing fluence rates of red light are exposed to blue light, the resulting blue 350 400 450 500 light–stimulated stomatal opening is linearly related Wavelength (nm) to the fluence rate of background red-light irradiation FIGURE 18.19 The absorption spectrum of zeaxanthin in (see the wild-type treatment in Figure 18.20) and to ethanol. 416 Chapter 18 FIGURE 18.20 Stomatal responses to blue light in the wild Wild type type and npq1, an Arabidopsis mutant that lacks zeaxanthin. Stomatal aperture (mm) 2.8 npq1 (mutant Stomata in detached epidermis were irradiated with red light for 2 hours, and 20 µmol m–2 s–1 of blue light was lacking zeaxanthin) added for one additional hour. Stomatal opening in the wild type is proportional to the fluence rates of background 2.4 red light. In contrast, npq1 stomata lacked this response and showed reduced opening under both blue and red light, probably mediated by guard cell photosynthesis. (From Frechilla et al. 1999.) 2.0 50 100 150 zeaxanthin content (Srivastava and Zeiger 1995b). Background red light (mmol m–2 s–1) The same relationship among background red light, zeaxanthin content, and blue-light sensitivity has been found in blue light–stimulated phototropism of enzyme that converts violaxanthin into zeaxanthin. corn coleoptiles (see Web Topic 18.4). The specificity of the inhibition of blue light–stimu- lated stomatal opening by DTT, and its concentration Blue light–stimulated stomatal opening is completely dependence, indicate that guard cell zeaxanthin is inhibited by 3 mM dithiothreitol (DTT), and the inhi- required for the stomatal response to blue light. bition is concentration dependent. Zeaxanthin forma- tion is blocked by DTT, a reducing agent that reduces In the facultative CAM species Mesembryanthemum S—S bonds to –SH groups and effectively inhibits the crystallinum (see Chapters 8 and 25), salt accumulation Light energy CHLOROPLAST (PAR) ADP ATP H+ + Pi CO2 sensing by rubisco ATP synthase Ribulose-1,5 CO2 biphosphate H+ Carboxylation Calvin Regeneration cycle ATP Grana H+ + thylakoid Reduction NADPH Triose phosphate ADP H+ ATP + Pi ADP + Pi Violaxanthin Blue-light + NADP+ sensing npq1 Zeaxanthin CYTOPLASM ? phot1 phot2 14-3-3 Serine/threonine H+ protein kinase H+ Cl– K+ P C terminus H+-ATPase Inactive H+ Cl– K+ FIGURE 18.21 A sensory transduction H+ cascade of blue light–stimulated stomatal Active opening.  Blue-Light Responses: Stomatal Movements and Morphogenesis 417 shifts its carbon metabolism from C3 to CAM mode. In the C3 mode, stomata accumulate zeaxanthin and SIGNAL TRANSDUCTION show a blue-light response. CAM induction inhibits Sensory transduction cascades for the blue-light responses the ability of guard cells to accumulate zeaxanthin, encompass the sequence of events linking the initial and to respond to blue light (Tallman et al. 1997). absorption of blue light by a chromophore and the final expression of a blue-light response, such as stomatal open- The blue-light response of the Arabidopsis mutant ing or phototropism. In this section we will discuss avail- npq1. The Arabidopsis mutant npq1 (nonphotochemical able information on signal transduction cascades for cryp- quenching), has a genetic lesion in the enzyme that con- tochromes, phototropin, and zeaxanthin. verts violaxanthin into zeaxanthin (see Figure 18.21) (Niyogi et al. 1998). Because of this mutation, n