1 GREEN LIGHT IN PHOTOMORPHOGENIC DEVELOPMENT By STEFANIE ANNE MARUHNICH A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007
2 2007 Stefanie Anne Maruhnich
3 To Elie and my Mom.
4 ACKNOWLEDGMENTS I thank Elie and my family for their support. I thank Elie for being a constant source of strength, my Mom for her support and patience, Stush for his ent husiasm and assistance and my Dad for his interest and advice. I thank Dr. Te rance Lucansky who introduced me to fundamental botany concepts, was my undergraduate advisor, and later a good friend. I thank Dr. Kevin Folta, my graduate advisor and friend, for his guidan ce and encouragement over the years we have worked together. I thank Dr. Harry Klee, Dr. Karen Koch, Dr. Bernard Hauser, and Dr. David Oppenheimer for participating on my committee. I appreciate all of your advice, support, and encouragement through this process. I thank Dr. Curt Hannah, Dr. Mark Se ttles, Dr. Chris Chase, and Dr. Maria Gallo for their assistance, and Andrea Eveland, Nicole Frederick, and Angie Lay for their friendship over the years.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF FIGURES................................................................................................................ .........7 LIST OF ABBREVIATIONS..........................................................................................................8 ABSTRACT....................................................................................................................... ............10 CHAPTER 1 INTRODUCTION..................................................................................................................12 2 LITERATURE REVIEW.......................................................................................................15 Introduction................................................................................................................... ..........15 Phytochromes and Cryptochromes are Green Light Receptors..............................................18 Early Green Light Effect s on Vegetative Growth..................................................................19 Green Light Affects Orga n Growth and Stature.....................................................................20 Green Light and Tropism........................................................................................................22 Heliochrome.................................................................................................................... .......24 Green Light Opposes Stomatal Opening................................................................................26 Green Light Effects on Leaf Grow th and Stomatal Conductance..........................................28 Early Stem Elongation.......................................................................................................... ..30 Green Light Down Regulates Plas tid Transcript Accumulation............................................31 A Connection to Plant Biomass..............................................................................................32 Cryptochrome-Dependent Green light Effects.......................................................................33 Is The Photosensor Class Complete?......................................................................................35 Conclusions.................................................................................................................... .........38 3 GREEN LIGHT EFFECTS ON EARLY DEVELPOPMENT..............................................42 Introduction................................................................................................................... ..........42 Results........................................................................................................................ .............45 Hypocotyl Elongation......................................................................................................45 Green-Light-Induced Hypocotyl El ongation is Dose-Dependent...................................46 Other Observations..........................................................................................................46 Anthocyanin and Chlorophyll Levels..............................................................................47 Photoreceptor Mutants.....................................................................................................47 Discussion..................................................................................................................... ..........48 Materials and Methods.......................................................................................................... .51 Plant Materials................................................................................................................ .51 Light Sources and Treatments.........................................................................................51 Hypocotyl Elongation Assays.........................................................................................52 Anthocyanin and Chlorophyll Levels..............................................................................53
6 4 GREEN LIGHT EFFECTS ON MATURE PLANTS............................................................59 Introduction................................................................................................................... ..........59 Results........................................................................................................................ .............61 Discussion..................................................................................................................... ..........68 Materials and Methods.......................................................................................................... .71 Plant Materials................................................................................................................ .71 Plant Growth................................................................................................................... .71 Light Sources.................................................................................................................. .72 Measurement...................................................................................................................72 Anthocyanin Accumulation.............................................................................................73 5 CANDIDATE GENE APPROACH TO IDENTIFY GREEN LIGHT PATHWAY COMPONENTS.....................................................................................................................81 Introduction................................................................................................................... ..........81 Results........................................................................................................................ .............83 Isolation and Characterization of T-DNA Mutants.........................................................83 Hypocotyl Elongation......................................................................................................83 Chloroplast Transcript Regulation..................................................................................84 Pharmacological Studies.................................................................................................84 Discussion..................................................................................................................... ..........85 Materials and Methods.......................................................................................................... .86 Plant Lines.................................................................................................................... ...86 Photomorphogenic Development....................................................................................87 Transcript Analysis..........................................................................................................87 Retinal Treatments...........................................................................................................87 6 FUTURE GREEN LIGHT RESEARCH................................................................................92 LIST OF REFERENCES............................................................................................................. ..94 BIOGRAPHICAL SKETCH.......................................................................................................102
7 LIST OF FIGURES Figure page 2-1. Quantum energy distribution of full sun light and under the shade of leaves (canopy shade)......................................................................................................................... ........39 2-2. Data reproduced from Wents green-depletion experiments in 1957................................40 2-3. The proposed photocycle for plant cryptochromes............................................................41 3-1. Supplemental green-light-induced hypoc otyl elongation is specific to green wavebands...................................................................................................................... ....54 3-2. Fluence rate/response experiment......................................................................................55 3-5. Hypocotyl elongation experime nts for photoreceptor mutants (Wild type [Col], in purple, cry1 cry2 mutants in blue, phyA phyB mutants in green). ....................................58 4-1. Supplemental green light induced a shade response..........................................................74 4-2. Supplemental green light induced petiole elongation and inhibited leaf expansion..........75 4-3. Supplemental green light effects ar e maintained in photoreceptor mutants. ....................76 44. Green light responses ar e fluence-rate-dependent.............................................................77 4-5. Anthocyanin accumulation under increasi ng amounts of supplemental green light.........78 4-6. Anthocyanin accumulation decreased under supplemental green light for wild type (Col) plants (purple) and cry1 cry2 mutants (green).........................................................79 4-7. Vegetative features of Fragaria vesca after 9 weeks growth under RB (grey columns) and RGB (purple columns) light environments. ...............................................80 5-1. Insertion sites for the ccd8 T-DNA lines obtained via the Salk Institute .........................88 5-2. The ccd8 mutants are defective for green -light-induced stem elongation.........................88 5-3. The ccd8 mutants demonstrate green-light-i nduced hypocotyl elongation in 2-d-old seedlings...................................................................................................................... .......89 5-4. The ccd8 mutants were aberrant for green -light-mediated down-regulation of chloroplast transcripts (psaA) but wild type for phy gene regulation................................90 5-5. Partial rescue of green-light-mediated dow n-regulation of chloroplast transcripts in ccd8 mutants with micromolar amounts of ATR...............................................................91
8 LIST OF ABBREVIATIONS LED Light-emitting diodes PAR Photosynthetically active radiation phy Phytochrome cry Cryptochrome nm Nanometer PPF Photosynthetic photon flux phot Phototropin LOV Light, oxygen, voltage domain FMN Flavin mononucleotide ABA Abscisic acid PFR Far-red-absorbing phytochrome PR Red-absorbing phytochrome FAD Flavin adenine dinucleotide R Red light B Blue light G Green light d Days h Hours P P-value A Absorbance g Grams Col Columbia L er Landsberg erectus MS Murashige and Skoog
9 HCl Hydrochloric acid mm Millimeter # Number diff Differentiated lvs Leaves obs. Observation cm Centimeter m Meter s Second C Celsius PCR Polymerase chain reaction min Minute KCl Potassium Chloride CaCl2 Calcium Chloride
10 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy GREEN LIGHT IN PHOTOMORPHOGENIC DEVELOPMENT By Stefanie Anne Maruhnich August 2007 Chair: Kevin Folta Major: Plant Molecula r and Cellular Biology Light quality, quantity, and durat ion provide essential environm ental cues that shape plant growth and development. Over the last century, re searchers have worked to discover how plants sense, integrate, and respond to red, blue, and fa r-red light. Green light is often considered a benign wavelength with little to no effect in plant development. However, sparse experiments in the literature demonstrate that green eff ects are often counterint uitive to normal light responses and oppose redand blue -light-induced responses. Green light effects on plant growth and development are described here through th e use of custom, tunable LED, light-emitting diode, chambers. These light sources allow for specific light qualities and quantities to be administered. The effects of green wave bands were assessed when red and blue photomorphogenic systems were active to answer th e question: Are the eff ects of an inhibitor (green light) more evident in the presen ce of inducers (red and blue light)? In seedlings, supplemental green light increased hypocotyl elongation opposite to classical inhibition of hypocotyl elongation as sociated with growth in light and induced by red and blue wavebands. Results indicate that added green light induced a revers ion of light-grown phenotypes. In mature plants, supplemental green light induced phenotypes typical of the shadeavoidance syndrome, including elongated petioles, smaller leaf areas, and leaf hyponasty. These
11 responses are typical of lower-light conditions or far-red enriched environments. Contrary to farred-light-induced shade-avoidance, data indica te green delays flower ing. In Arabidopsis and strawberry plants, anthocyanin le vels also decreased when green light was added to red and blue light treatments, which is again opposite to normal light-induced phenotypes. Photoreceptor mutants were tested and indica te green light effects in early development are cryptochromedependent. However, green-light-induced shad e-avoidance responses were cryptochromeindependent. A candidate gene approach was used to identify other elemen ts required for green light sensing and/or response. Defects in some green light responses were observed for mutants in CCD8/Max4, a putative carotenoid cleavage enzyme with high sequence simila rity to a critical enzyme in animal vision. These data support a ro le for green light in plant development which opposes normal light-induced responses and indicat e the existence of at least two green light sensing systems.
12 CHAPTER 1 INTRODUCTION Light quality, quantity, and durat ion provide essential environm ental cues that guide plant growth and development. The effects of red, far-re d, and blue light have b een well-characterized. Recent work demonstrates that green light, histori cally considered ineffective relative to potent far-red, red, and blue wavebands, plays an important role in seedling development which opposes normal photomorphogenic growth (redand blue-light-induced responses). Green light effects are often subtle and examined under monochromatic green light regimes. These experiments were designed to test the effects of an apparent inhibito r (green light) in the presence of inducers (red and blue light) of typi cal light responses. Perhaps one reason that green light effects are under-represented in the literature is due to the fact that it is difficult to observe the effects of an inhibitor when forward-acting sy stems are inactive. For these experiments, light emitting diodes (LED), which provide narrow-bandwidth light, were used to test the effects of green light in the presence of red and blue light on the growth of Arabidopsis thaliana plants. Green-light-induced effects were observed unde r combinatorial light regimes in newly germinated seedlings as well as mature plants. In early development, the addition of green light resulted in longer hypocotyls. These green light effects were induced by low fluence green light but inhibited as fluence rates of green increased. The responses were also cryptochromedependent. Mature plants grown under red and blue light (RB) treatments displayed characte ristics typical of normal photomorphogenic growth, i.e. short overall plant stature, open and expanded leaf blades, and leaves oriented relatively parallel to soil. When green light was added to the red and blue light background (RB+G), total light quantity was increased, and plants disp layed characteristics a ssociated with shadeavoidance syndrome, i.e. increase d petiole elongation, increased l eaf angle, and decreased leaf
13 area. Here increased total fluence (RB+G compar ed to RB) caused phenotypes associated with lower-light phenotypes, and as gr een light was added to the red and blue light background these lower-light associated phenotypes became more apparent. Shade-avoidance responses are also induced by far-red light enrichment (or low ratios of red to far-red), low blue light, and low photosynthetically active radiati on (PAR). There was very little to no far-red light (<.0001) present under these treatments. Also, blue light quantities were the same between treatments (RB and RB+G) and plants grown under RB exhib ited characteristics typical of normal photomorphogenic growth. PAR was increased in RB+G treatments compared to RB, while plants grown under RB+G displayed characteristic s associated with shade-avoidance responses and plants grown under RB did not. Therefore, th e shade-avoidance phenotypes seen in plants grown under RB+G can only be attri butable to green wavebands. Although most green light effects in mature pl ants mimicked shade-avoidance responses, preliminary data show that supplemental green li ght delayed flowering. Th is delay of flowering is opposite to the promotion of flowering de monstrated by the shade-avoidance syndrome associated with low ratios of red to far-red wavelengths. These results indicate a point of divergence between far-reda nd green-light-induced shade-a voidance syndromes and suggest the existence of a novel green se nsing system. Support for this novel sensing system was that these phenotypes persisted in all pho tosensory mutants tested, including phyA phyB cry1 cry2 and phot1 phot2 mutants. In addition to this, a further candidate gene approach was taken in which a gene with high sequence similarity to a visual cycle enzyme, Ccd8 was found to be required for green-lightinduced down-regulation of plastid transcripts early in seedling development.
14 The aim of this project was to characterize the role of green light in plant development and to establish tools to elucidate components of the green light sensing pathway(s). Little is known about green lights role in plant developmen t thus far, but experi ments in the literature contribute to a common theme-green light responses seem to work against or antagonize normal light-induced phenotypes, such as redand blue-light-induced effects. The results support this theme and increase our understanding of how plants sens e and respond to their light environment. In addition, this work demonstrat es the effectiveness of using LED-based arrays and provides the tools necessary to expand this work to identify other green light pathway components. These tools can also be used to as sess green light effects on other plant species. If green light negatively affects a quality of intere st in a given species, ma nipulation of the light environment through colored mulch in the field or colored filters within greenhouses may lead to increased crop production and/or commerc ial plant production in greenhouses.
15 CHAPTER 2 LITERATURE REVIEW Introduction Over a century of photomorphogenic research de scribes how light plays an important role as an environmental signal in addition to its ro les in photosynthesis. Experiments have identified and described the effects of red, blue, a nd far-red light signaling pathways. Although, new components are discovered, the complex pathways that transduce thes e light signals are generally well-defined. A less-studied, but equa lly important waveband imparting environmental information is green light. A complete story of how plants sense and respond to green wavebands, or even a full range of green-light-in ducible responses is not yet available. However, this review of the literature de scribes and examines experiments that indicate a role for green light in shaping plant growth and development. One general idea presented herein is th at although red and blue light promote photomorphogenesis, green wavebands tend to have opposite effects and temper responses to red and blue light. A common theme in biology is th at organisms employ opposing systems to tightly monitor, adjust, and constrain developmental progr ams, and a negative influence of a green light sensory system would be consistent with this idea. Examination of the literature surrounding light mediated plant developmental research reve als several reports that support this view. For the purposes of this literature re view, green light is expanded to represent the green and yellow portions of the spectrum (500-600 nm). Green wave bands have been traditionally considered as developmentally inconsequential out side of their partial forward st imulation of red and blue light responses (phytochromes [phys] and cryptochromes [crys] can absorb green light, although inefficiently). However, there are many exampl es in the literature where monochromatic or
16 broadband green light treatments elicit effects on plant growth and development that do not conveniently fit w ith known light sensory systems. The accepted dogma in light biology is that all wavebands of visible light promote photomorphogenesis. However, evidence indicates that wavebands in the far-red region of the electromagnetic spectrum counter typical light-induced plant growth in some contexts. Borthwick et al. (1952) illustrated how far-red wa vebands, inefficient for photosynthesis, impart important environmental information. Generall y, far-red light counters the developmental processes initiated by red light, and the ratio of red to far-re d wavelengths dictate the activity of molecular, biochemical, and mor phological processes (Quail, 2002; De vlin et al., 2003; Chen et al., 2004; Casal and Yanovsky, 2005). This is a pr ime example of how wavebands with little impact on plant metabolism can function as a cue for adjustment of plant form and composition. These modifications may conserve valuable reso urces and provide increased fitness to plants growing in lower-light environments. Green light is typically enriched in natural environments that are also enriched for far-red light, i.e. any environment that is covered by overhanging foliage such as the understory of a plant canopy or in a densely-packed field. Therefore, a logical hypothesis is that green light may indu ce similar responses as far-red light. This chapter presents evidence that green light responses oppose t hose of red and blue light, and are at times similar to those of far-red wavebands. Although the green signaling components are largely unknown, this work demons trates a role for green wavebands in plant growth and development. However, interpretation of classical work presents some difficulties for the following reasons: 1. Experiments were us ually performed under br oadband light conditions not exclusively emitting green light 2. Some studies measured light in foot-candles or ergs, 3. Light treatments were not equalized across the spectrum, and/or 4. Researchers used
17 combinations of light treatments that obscured simple interpretation. However, some of these experiments did indicate that gr een wavebands cause a specific set of responses not easily attributable to red, far-red, or blue light and their receptors. Th e elucidation and characterization of genetic elements in Arabidopsis thaliana that transduce red, far-red, and blue signals and their corresponding mutants now provides tools that allow green light responses to be studied in isolation from other light sensory inputs. Many of the responses induced from the green light portion of the spectrum are counterintuitive, ofte n opposing typical light e ffects (Klein et al., 1965; Ahmad et al., 1998; Frechilla et al., 2000; Talbott, 2002; Ei singer et al., 2003; Folta, 2004; Dhingra et al., 2006; Bo uly et al., 2007). There are many examples in natural environm ents where green light is enriched, thus emphasizing the importance of exploring the way in which plants sense and respond to these wavebands. For example, under the cover of leav es, whether in the understory of a plant canopy or the shade of neighbor plants in a densely-packed field, plants are subjected to a pronounced contrast to the unfiltered solar illumination. Primarily, there is a decrease in radiant flux and a shift in the ratio of visible to far-red wavelengths (Figure 2-1) Although red and blue wavebands are absorbed by overhanging foliage, far-red wavebands are transmitted through leaves and enriched in the understory. In addi tion to far-red enrichment, ratios of red and blue to green light shift towards green light, since gr een light is readily reflected from plants as well as being transmitted through them. Green light moves effici ently through the plant body, playing more of a role in photosynthesis than red or blue light in some contexts (Sun et al., 1998), suggesting that green light may prove useful as a signal to tissues not directly e xposed to the light environment. Potential green light effects may also vary w ith developmental context, since an etiolated
18 seedling emerging from the soil has negligible ch lorophyll and will allow gr een light to penetrate as efficiently as blue, red, and far-red light. Phytochromes and Cryptochromes are Green Light Receptors Phytochromes and cryptochromes absorb and re spond to green light. Th is is likely a major contributor to the lack of exhaustive studies on green light. Although phytochromes are principally regarded as red/far-red reversible pi gments, they absorb well in the blue portion of the spectrum and are extremely sensitive to all light qualities, especially in dark-grown seedlings where light labile phyA is abundant (Goto et al., 1993). In addition to being sensitive to all light qualities, these receptors will initia te responses to low levels of light. In Arabidopsis, green light stimulates germination effectively through phyA and phyB systems (Shinomura et al., 1996). Green light establishes an activ e phy pool and even the most miniscule safelight green light treatments activate robust plan t responses (Mandoli and Briggs 1981; Steinitz et al., 1985; Dhingra et al., 2006). The cryptochromes regulate plant responses to blue and UV-A light (Lin, 2002; Spalding and Folta, 2005), and recent evidence indicates bi ological activity of a green-light-sensing state (Banerjee et al., 2007; Bouly et al., 2007). Ma lhotra et al., (1995) showed that these chromoproteins contain a flavin and a pterin as the light-excitable moieties. The presence of both the flavin and the pterin indicated that the cryptoc hromes rely on intramolecular electron transfer as part of their signaling m echanism (Malhotra et al., 1995). Lin et al. (1995) overexpressed Arabidopsis CRY1 in transgenic tobacco. The over expressors exhibited hypersensitivity not only to blue, but also to broadband green light, indicating that cryptochromes could direct stem growth inhibition even when stimulatory wavele ngths were red-shifted. However, recent reports by Bouly et al. (2007) and Banerj ee et al. (2007) show that the green-light-absorbing state of cry1 and cry2 reverses blue-light-induced response s. These findings are disc ussed in detail in the
19 final sections of this chapter. In addition, at least one additional li ght sensor system, or previously undefined aspect of a known system mediates speci fic effects of green wavebands. Here too, green responses tend to arrest or at tenuate the physiological phenotypes associated with normal photomorphogenic progression. Ther efore, green light responses can be characterized as those that are cryptochrome dependent and those that are cryptochrome independent Clearly, the phytochromes and cryptochromes can both function as sensitive green light photoreceptors in plants, but the efficiency of th ese systems in processing the green light signal is poor relative to their capacity to respond to red and blue wavebands. With this in mind, green light effects could be the result of low-level coaction between red and blue sensory systems, as outputs from minimal phy and cry activation may pr esent what mistakenly appear to be greenlight specific phenotypes. This possi bility has likely led to deprio ritization of research on green light signaling pathways. However, current res earchers possess genetic and physiological tools that allow green light effects to be dissected from those induced by developmentally dominant wavelengths, red and blue. Other tools that ma ke elucidation of green light responses and pathways possible are the availability of high power, narrow bandwidth li ght sources, access to double/triple photoreceptor mutants, and growth assays with great sensitivity. Together these tools have been used to characterize the often subtle effects of green illumination. Early Green Light Effects on Vegetative Growth In his book Experimental Control of Plant Growth, Frits Went (1957) examined the effects of light quality on seed ling growth by assessing tomato ( Lycopersicon esculentum ) seedling dry weight. The results of these experi ments have been reproduced in Figure 2-2 and illustrate that seedlings grown under red and blue light produce more vegetative tissue than those grown under the same fluence rate of white light (consisting primarily of red, blue, and green
20 wavebands). This result indicates that the added green light com ponent has a negative effect on seedling growth in terms of dry weight; however, green light in these experiments was added at the expense of red and blue light. Since red and blue are known to promote photomorphogenic development (in this case, increased seedling dry weight), lower seedling mass for plants grown under white light is expected due to decreased red and bl ue light. However, further experimentation led to the discovery that as fluence rates increased, plants grown under white light acquired a given mass before reaching a plateau, whereas plants grown under conditions where green light was reduced achie ved a higher dry weight at satura tion. In other words, plants grown under reduced green light were able to reach greater dry weights before reaching a plateau, regardless of how much fluence rates we re increased. This result indicates a negative role for green light in photomorphogenic develo pment, illustrated in this case by the maximal vegetative growth that may be achieved. Green Light Affects Organ Growth and Stature Similar results were found by Klein and coll eagues in the 1960s when they discovered a negative effect of green wavebands on tissue culture growth. In these experiments, tissue culture growth was examined under depleted and supplem ental green light conditions. Taking Wents experiments one step further, th e effects of various light qual ities on growth inhibition were examined and Klein found that the most delete rious wavelengths peaked at 550 nm (Klein, 1964). Although this work demonstrated a negative ro le for green light in tissue growth, Klein later extended this work to mature plants and observed additional e ffects of green light on marigold ( Tagetes erecta L .), tomato, and impatiens ( Impatiens balsamina L .) (Klein et al., 1965). For reduced green light treatments, lavender filters were used toge ther with fluorescent and incandescent bulbs to effectively reduce th e level of green light. For supplemental green
21 treatments, these wavelengths were added to a background of white light. Results were complementary to Wents data (1957) and indicat ed that, when grown under green-light-depleted conditions, marigold height, fresh weight, and dry weight increased 30-50% over full-spectrum treatment. The authors interpreted these data to mean that removal of green light enhanced plant growth. However, this interpretation is complicated by the fact that lavender filters effectively reduce blue light as well as gr een light. Therefore, enhanced gr owth seen under reduced green light may have resulted from reduced photosynt hetically active radiation (PAR) (400-700 nm). Here, lower PAR may be responsible for taller pl ants since red and blue wavebands inhibit stem elongation. Total photosynthetic pho ton flux (PPF) was only record ed for white light sources, which were 1000 and 500 footcandles (approximately 200 and 100 moles m-2 s-1), and not under lavender filters. The lack of recorded PPF for all treatments leads to difficulties in interpretation of these data. More definitive conclusions can be drawn from experiments in which green light was added to a background of white light. Under conditions of enhanced green light, plants were shorter and had lower ratios of dry to fresh wei ght. In these experiments supplemental green light treatments apparently represent greater fluence rates when compar ed to white light alone, since green light was added to the wh ite light background. Data showed that increased fluence rates under white plus green light led to less vegetative growth than white light alone. These results are consistent with those of Went (1957) and recent studies by Dougher and Bugbee (2001). Dougher and Bugbee (2001) compared the growth of lettuce ( Lactuca sativa ) seedlings under metal halide and high pressure sodium lamp s. The authors were careful to control for phytochrome equilibrium, quantities of blue, red, and far-red light, and relative ratios between them. The only difference between treatments wa s the green light component (580600 nm).
22 Therefore, when data indicated differences in dry mass, leaf area, and chlorophyll content, meaningful conclusions could be drawn. Trends ag reed with previously documented findings-green light has an inhibitory effect on plant growth. Green Light and Tropism Blue light is known to induce plant movement s such as the bending of some structures towards light and the relocation of chloroplas ts, and the responsible photoreceptors are the phototropins (Briggs and Christie, 2002; Spalding and Folta, 2005). In addition to blue light, green also stimulates phototropic responses (Stein itz et al., 1985). Experiments demonstrated that green light would promote bendi ng towards light but required more time and 10-fold greater fluence rates to generate an equi valent response to that of blue light (Steinitz et al., 1985). In addition to defining a role for green light in this response, the aut hors argued that green wavelengths were acting through the blue light receptor, whic h was later identified as phot1 (Steinitz et al., 1985). Th e green-light role was later verified when phot1 mutants failed to demonstrate wild-type phototropic responses to green wavelengths (L iscum and Briggs, 1996). When an action spectrum was examined, gree n-light-induced phototropic responses were found to be more efficient as wavelengths approached the blue portion of the spectrum, i.e. shorter wavebands of green light were more effective in stimulating this response (Stenitz et al. 1985). Whether these data are the result of phot sensitiv ity or blue contaminatio n of green light sources is unknown, but results indicate that phots are green light receptors in addition to crys and phys. Later analysis of photocyc ling by the oat phot1 LOV2 domain indi cated that blue light activation drives formation of a greenand red-light-abs orbing flavin mononucleotid e (FMN) triplet state that is extremely transient (on the order of nanoseconds) (Swartz et al ., 2001; Kennis et al., 2003). Due to their short-lived na ture, it is unlik ely that these species would play any kind of meaningful role in phototropin act ivity. The work by Steinitz et al. (1985) that elucidated an
23 action spectrum for green-light-induced photot ropic bending utilized carefully-defined illumination conditions that combined narrow-bandpa ss filters with sharp cut-off filters that resulted in little to no transmission below 500 nm. Therefore, when data indicated considerable differences in dry mass, leaf area, and chlo rophyll content, they could draw significant conclusions. Trends agreed with previously doc umented findings-green light has an inhibitory effect on plant growth. In addition to a possible role in the bendi ng of some plant parts towards light, green wavelengths have been implicated in root tropisms (Klein, 1979). In these experiments, root growth habits of curly cress ( Lepidium sativum ) were examined. Phytochromes are known inducers of diageotropic root growth. In curly cress, however, positive gravitropic responses are not phytochrome-dependent. This allows for exam ination of green light effects on root growth independent of phy involvement. Data indicated that green light sl ows geotropic root curvature. Examination of specific green wa velengths indicated that the response was most effective when 546 nm light was administered, whereas wavelength s shorter than 520 nm or greater than 580 nm had no effect on slowing normal root responses. Th ese experiments identified the most effective wavebands for this green light response and st rengthened the link between green wavebands and root tropic responses. Interestingl y, this work also demonstrated a reversal of green-light-induced inhibition of root growth by ot her light qualities. Here, waveba nds distributed at 620 nm were most effective at reversing green light effects, whereas irradiation near 660 nm (phytochromes peak absorption) had no effect. In addition to red light responses, blue wavelength effects were observed, and demonstrated that blue did not re verse green light responses. These results are consistent with a cry-independent mechanism.
24 In addition to stem and root tropic responses, green wavelengths have been associated with leaf movements. Leaf inclination has been linked to phytochrome induction in environments with low ratios of red to far-red. Studies by Mull en et al. (2006) demons trated that changes in leaf angle required phyA, B, and E. Interestingl y, the green light component of white light induced the response in hy1 and phyA phyB phyE mutants, indicating th at although phyA, B, and E were required for low red: far-red induction of leaf inclination, they were not required for green-light-induced changes in l eaf inclination. These experime nts demonstrated that although green light induced changes in leaf position that were similar to those in low red: far-red environments, the wavebands operated via two separa te light sensing systems. In addition to the phy mutants, npq1 mutants were examined, since the la tter are required for green-light-induced reversal of blue-induced stom atal opening (Frechilla et al ., 2000; detailed below). The npq1 mutants showed green-light-induced leaf inclinat ion similar to that of wild type. Results indicated that NPQ1 was not required for this green light response and provided a precedent for the idea that there could be multiple green sens ors involved in green light responses. In addition to npq1 the npq2 mutants with lesions in a gene requi red for ABA synthesis were unable to induce the leaf-inclination res ponse. Application of ABA restor ed normal leaf angles, so a change in ABA levels may be one result of green li ght responses. Heliochrome Work described in the previous section dem onstrated that green light effects on leaf inclination mimicked those of shade environments with a low red to farred ratios. Experiments by Tanada also examined interactions between gr een and far-red light. The results of this and later work led to the suggestion of a far-red/gree n light reversible receptor. The author later coined the term heliochrome since this appe ared to act similarly to phytochrome. Initial experiments were based on the closing of Albizzia julibrissin pinnules (Tanada, 1982). Albizzia
25 julibrissin pinnules exhibit nyctinastic closure whic h is delayed by far-red light 710-730 nm. This delay, which requires substant ial fluence rates (18-43 mol m-2 s-1) of far-red light was completely negated by co-illumination with dim green light (0.01-5 mol m-2 s-1). The pinnules were also examined in experiments similar to those of Borthwicks early work (1952) describing phy toggling after red and far-red light applicatio n. The pinnules demonstrated a similar toggling response to green and far-red light treatments, whereas red light had no effect (Tanada, 1982). These experiments indicated the existence of a green light/far-red reversible sensor. In addition to green light, blue was found to s timulate reversal of th e far-red-induced delay in pinnule opening (Tanada, 1984). Green light coul d completely negate the blue light effect. Tanada concluded that the far-red absorption state of heliochrome was sensitive to blue as well as green light. A similar connection between far-red and green light was reported in Brassica campestris (Tanada, 1984). In this species fa r-red light induces prol ific flowering when given as an end-ofday pulse. As in the pinnule experiments, gree n light (550 nm) given at low fluence rates was able to effectively reverse far-red (710 nm) induc ed flowering effects, whereas red light could not. Similar to pinnule opening, th is response exhibited toggling e ffects like those observed for red, far-red toggling by Borthwick (1952). Together these studies describe green light responses that actively reverse far-red-lightinduced responses. In 1997, Tanada proposed that heliochrome was a heme-based receptor that toggled between far-red and green light sensing states. Tanadas work illustrates another example from the literature where green light effects cannot be easil y reconciled with known photoreceptors and implies the existe nce of a novel green light sensor.
26 Green Light Opposes Stomatal Opening Zeiger and colleagues (Frechilla et al., 2000) demonstrated that a brief pulse of green light could prevent blue-light -mediated stomatal opening in Vicia faba epidermal peels. Closer evaluation of this phenomenon revealed a blue-green reversible dichromaticity, as the quality of the last pulse of light dictated the physiological response obser ved. If green light was followed by blue, then stomates opened, whereas, if the pu lsed light sequence was green, blue, green, the stomates remained closed. These researchers went on to show that the unusual stomatal response was dose-dependent with the most significant eff ect occurring at a ratio of 2:1 for green:blue (Talbott et al. 2002). An action spectrum revealed 540 nm light to be the most effective wavelength for reversal (Frechilla et al. 2000). In addition, this re sponse persisted in a background of red light, indicating that the blue-green pathway wa s separate from previously described phytochrome or photosynt hesis-driven pathways that influence stomatal aperture. Later Talbott et al. (2002) observe d this blue-green effect in a diverse suite of plant species, demonstrating that the effect was evident throughout the plant kingdom. The authors suggested that th e molecular entity mediating the absorption and response to blue and green light may toggle between active a nd inactive states simila r to the phytochrome PFR and PR states (Frechilla et al., 2000). One candida te put forth was a carotenoid, zeaxanthin, which was previously described as a candidate for blue-light-induced stomatal opening (Frechilla et al., 1999). The action spectrum for this particular response (green-light-induced stomatal closure) matched the absorption spectrum for a carotenoid, but was red-shifted by 50 nm. The results were confirmed by Eisinger et al. (2003) who showed that a green-light pulse could negate the effect of ultravio let light on stomatal opening. Later the phototropins were implicated in the co ntrol of stomatal apertu re (Kinoshita et al., 2001). In combination with the studies by Zeig ers group, this finding suggested that the
27 phototropins may be blue-green reversible, ye t analyses of the phototropin LOV (light, oxygen, voltage) domain did not support th is hypothesis. Absorbance in th e green was found to be highly transient (on the order of a few nanoseconds) (Swartz et al., 2001; Kennis et al., 2003) This discrepancy was addressed by Talbott et al. (2003) with a genetic study in Arabidopsis mutants. Mutants analyzed were phot1 phot2 double mutants and npq1 mutants, that had a lesion in violaxanthin de-epoxidase, thus minimizing zeaxanthin accumulation (Niyogi et al., 1998). Results revealed that NPQ1, but not ph ot1 or phot2, was required for the blue-green stomatal response pathway. In this study, the aut hors examined various mutants together with redand blue-light-induction of stomatal opening, and the pot ential far-red/green light reversibility of the responses. The phot1 phot2 and npq1 mutants behaved like wild type plants in terms of the red/far-red stomatal response. Furthe r investigation demonstrated viable red/far-red and blue-green light reversibil ity for stomatal responses of phot1 phot2 double mutants, however these mutants required higher fluence rates of li ght for blue-induction of stomatal opening. These data confirm that phot1 and/or phot2 regulate the blue light stom atal response; however they do not appear to be the photoreceptors mediating the blue-green light reversible facet of stomatal opening. Interestingly, npq1 mutants lacked the capacity to re spond to blue light, but maintained red-induction and far-red reversal. These tests demonstrated the existence of independent stomatal regulatory pathways, defined discrete roles for NPQ1, phot1, an d phot2 in the response, and further illustrated th e role of NPQ1 in blue-green revers ible guard cell action (Talbott et al., 2003). Later work by this group showed that green light effects involved a circadian component that was most prevalent in the morning (T albott et al., 2006). Th is study supports the involvement of NPQ1 in the blue-g reen stomatal pathway, since the npq1 mutant did not respond
28 to blue or green light. However, phot1 phot2 double mutants and wild type plants showed similar responses. This work, in conjunction with other anal yses, indicated that the effects of green light were conditional and that the use of mutants and specific light treatments was required to delineate specific pathways. An a dditional report demonstrated a ro le for cryptochromes in guard cells, and indicates that this system was distinct from that mediating phot effects (Mao et al., 2005). Recent research shows that cryptochromes undergo toggling fr om active to inactive states depending on green and blue light (Bouly et al., 2007). Therefore, it will be important to test if cryptochromes are relevant to the blue-g reen reversibility of stomatal opening. Green Light Effects on Leaf Gr owth and Stomatal Conductance The effects of green light on stomatal ope ning noted by Zeigers group were extended to whole plants by NASA scientists. Plant growth in artificial environments remains a key provision to long-term space colonization. Therefore, NASA scientists have explored the effects of combinatorial light conditions on plants. Many of these studies simply focused on the effects of narrow-bandwidth red and blue sources comp ared to conventional sources (Brown et al., 1995; Goins et al., 1997; Yorio et al., 2001). On e concern emerged when plants were grown under certain light conditions. Plants appeared black or purple when grown under red and blue LEDs. This rendered it difficult for the potential crew to monitor plant growth and health, and also, miscolored plants were not as visually appealing (Kim et al., 2004). With the goal of making plants appear gr een, NASA scientists assessed the effects of green light supplementation to a red and blue ba ckground. The result was that addition of this allegedly benign wavelength genera ted conspicuous effects. The e xperiments differed from those performed by Went and Klein in that in th ese experiments PPF was kept constant and the proportion of added green light was varied. This approach ha d the advantage of keeping metabolism static, yet the disa dvantage of skewing activati on of photosensory networks
29 contributing to developmental responses. Thes e studies also used different species and developmental states relative to earlier studies. For this reason the results must be considered independently of the prev iously described work. In these reports, the effects of combinatoria l red, blue, and green ( RB+G) light treatments on leaf growth and stomatal conductance in lettuce were compared to red and blue (RB) alone (Kim et al., 2004, 2004). Green light supplied by green fluorescent lamps was added to a background of red and blue LED light. There was very little (if any) far-red light in these experiments, which is important for discountin g potential phytochrome interpretations. The authors discovered that lettuce plants grown in RB+G treatments produced larger, thinner leaves that had higher specific leaf area when comp ared to those grown with RB alone (Kim et al., 2004). Also, plants grown under RB treatments de monstrated higher stomatal conductance when compared to those under RB+G, with the lowest st omatal conductance repo rted in plants grown under green fluorescent lamps alone (Kim et al., 2004). Additionally, while stomatal conductance was greater under cool white fluorescent lights than in RB+G treatments, the dry mass of the plants was greater in the latter. This result implies that weaker stomatal conductance did not negatively affect carbon assimilation (K im et al., 2004). Plant dry mass was greatest under RB+G treatments (where 24% of the spectrum was broadband green light) when compared to RB, the opposite of the effects noted by Went (1957; Figure 2-2). However, these results do agree with previous findings that plants grown in RB+G treatments had larger specific leaf areas than those grown under RB treatments (Kim et al., 2004). These experiments demonstrate that supplemental green light affects plant physiology in conditions where red and blue systems are saturated. It remains to be seen whether these effects are cry-de pendent or cry-independent, as they were performed in species where phot oreceptor mutants are not yet available.
30 Early Stem Elongation Once blue-green reversibility was identified in plants, namely in stomatal aperture pathways, experiments to examine green light eff ects in early development were initiated (Parks et al., 2001). For these studies, hypocotyl elongati on rate was examined in 2-d-old dark-grown seedlings. This organ represents a highly sensi tive system in terms of light response. For example, previous work using high-resolution image capture techniques were employed with Arabidopsis mutants to identify discrete ro les of phytochromes (P arks and Spalding, 1999), cryptochromes (Folta and Spaldi ng, 2001) and phototropins (Folta et al., 2003) in acclimation to the early light environment. In these experiments, dark-grown seedlings were exposed to a pulse of blue light (a strong inhibitor of hypocotyl elongation) followed by a pulse of green light. A characteristic phot response was observed in which seedlings exhibite d a normal inhibition in their first-phase of growth (Folta and Spalding, 2001; Folta et al., 20 03). However, within minutes, and only after receiving a green light pulse, seedling growth accelerated to 150% of the rate observed in darkness. The effect of green light was unlike an y previously described, and was also opposite to the accepted dogma that light i nhibits hypocotyl elongation and that growth rate is maximal in darkness. This unusual green-light-induced increa se in stem elongation rate was subsequently examined in greater detail. Sing le, etiolated seedlings were test ed for the elongation response to a brief green light pulse. Within minutes of a di m-green-safelight-quality light pulse, the dark grown seedling elongated faster th an it had in complete darkne ss (Folta, 2004). The response was dose-dependent, obeyed the Bunsen-Roscoe Law of Reciprocity, and was observed in response to a pulse barely detectable by eye. Green-light -pulse-induced growth accel eration was transient, with growth rates declining af ter an hour to those exhibi ted by dark-grown seedlings. Interestingly, green-light-mediate d growth induction persisted in cry phy phot and npq1
31 mutants, indicating either that the response was mediated by redundant function between known receptor classes or that it was initiated by a novel light sensor. The response also persisted in a background of dim red light, suggesting that phyto chrome was not required for this response. These data indicate that these gr een light responses are not initi ated by the major red and blue sensors, and do not require NPQ1, a zeaxanthin synthesis enzyme that regulates stomatal aperture (Frechilla et al., 1999). Similar studies of early seedling establishment attributed the long-term blue-green reversibility to cry recep tors (Bouly et al., 2007) and will be discussed further below. Together these studies delineate cry-independent and cry-dependent mechanisms associated with stem elongation and acclimation to the light environment. They also provide more evidence of green wavebands acting in opposition to red and blue wavebands. Green Light Down Regulates Plastid Transcript Accumulation Stem elongation experiments s uggest that green light impart s developmentally-influential information to seedlings; in addition it defines a ti me point at which to explore green light effects on the transcriptome of a developing seedling. Since stem elongation was induced by a brief pulse of green light and was well-established, if not complete, at 60 min (Folta 2004), gene expression was analyzed at this specific time point. Microarray experiments were performed in which 2-d-old dark-grown seedlings were expose d to a brief pulse of green light, or dark. The data presented both an ticipated and unexpected result s (Dhingra et al., 2006). For instance, a suite of genes known to be controlled by phyA (including Hy5 Pks1 and ELIP ) was induced, as though the plants were illuminated with red or far-red light (as in Tepperman et al., 2001). This result was predicted and also dem onstrated the phytochrome system was responding correctly. Although these phytochrome-induced tran scripts increased, a nu mber of chloroplast transcripts (including several previously show n as light-inducible) shar ply decreased after a pulse of green light. Examination of candidate transcripts showed that the response was rapid
32 (occurring within 15-30 min), sensitive (with a threshold <101 mol m-2) and obeyed the Bunsen-Roscoe Law of Reciprocity. Parallel eff ects of green light were observed in tobacco ( Nicotiana tabbacum ), indicating that this response was not confined to Arabidopsis (Dhingra et al., 2006). Similar to early gr een-light-induced stem elongati on, green-light-induced downregulation of plastid transcript s persisted in all photomorphoge nic mutant backgrounds tested. This suggests that the green light signal is lik ely dependent upon a novel se nsory system. Both of these green light responses (the increased st em elongation and the down-regulation of lightinduced chloroplast transcripts) are consistent with previous work. Both of these responses suggest that green wavebands pr ovide cues that indicate a su boptimal light environment for seedlings, and thus lead to a less advanced photomorphogenic habit. Responses to green light may allow seedlings to conserve valuable resources and direct their elongation beyond competing seedlings to regions of increased photosynthetic efficiency. A Connection to Plant Biomass Sommer and Franke (2006) presented eviden ce that the treatment of seeds with green lasers could lead to enhanced fr esh weight of plants at the time of harvest. Previous work by these authors examined laser li ght effects in speeding wound h ealing (Sommer et al., 2001) and increasing cell vitality in an imals (Sommer et al., 2002). They subsequently expanded their examination of high-fluence-rate light effects to plants, and f ound that dry carrot, radish, and cress seeds treated with a green light laser or intense green light LEDs produced plants with significantly greater biomass than control plants ( no light pretreatment). A ll plants shared equal conditions after the seeds were irradiated. All seedlings emerge d at the same time, so the differences observed later could not be simply attributed to phytochrome-induced enhancement of germination in laser-treated s eeds. Mature radishes and carrot s from laser treated seeds were twice as large as controls (Sommer and Franke, 2006).
33 While interesting, statistical rigor was thin in these experiments, and the authors also did not account for the possible explan ation that phytochrome would have been activated by their treatment, and this could have led to an advanced developmental state of the irradiated seedlings. With a resultant head start, seedlings might esta blish faster and more co mpletely than their nonirradiated siblings. However, follow up experiment s compared red to gree n light laser treatment of Arabidopsis seeds. Green light treated seeds germinated and emerged later than red treated seeds, yet still exhibited a larger end-point ro ot phenotype, suggesting that the pre-illumination did not drive a phy-induced enhancement of early development (A. Sommer communication with K.Folta). Cryptochrome-Dependent Green light Effects Cryptochromes have been implicated in gr een-light-induced responses. To understand cry involvement in these responses, one must firs t understand cry structur e. In 1995 two reports described association between cry1 and two poten tial chromophores, flavin adenine dinucleotide (FAD) and a second chromophore, 5,10-methylenyl te trahydrofolate (Lin et al., 1995; Malhotra et al., 1995). Studies in plants and fungi since the late 1960s suggested that flavoreceptor signaling would involve changes in the redox state of the chromophore. This possibility has since been confirmed, since a signifi cant amount of cryptochrome produ ced in insect cells was found to exist in the semi-reduced form (Lin et al., 1995). Biological consequences of the flavosemiquinone have been identified in Phycomyces (Galland and Tolle, 2003) and Arabidopsis (Bouly et al., 2007). Figure 2-3 depicts a model of the redox states of the flavin chromophore and how they relate to photosensor activity. Dark-grown plants contain the fully oxidized chromophore (FAD) that upon blue irra diation will convert to a semi-reduced (FADH) or fully reduced (FADH-) chromophore. The semi-reduced form is the biologically active, yet green light absorbing. Addition of green light driv es full reduction and inactivation (Bouly et al.,
34 2007). These chromophores likely undergo toggli ng similar to red/far-red toggling observed by Borthwick (1952). The degree of biol ogical activity is determined by the relative quanta of blue and green light, and resulting pools of semi -reduced, fully-reduced, and fully-oxidized chromoproteins. This theoretical model is supported by new bi ological evidence. Studi es of cryptochromes in insect cells demonstrated th at the initial light reactions in cryptochrome signaling depend on electron transfer from conserved tryptophan or tyrosine residues to reduce a flavin chromophore (Giovani et al., 2003). This is as predicted by m odels derived from photolyases (except that the latter possess the reduced flavin as a chro mophore). The action spectrum for cry1-mediated inhibition of hypocotyl elongation shows a peak at 450 nm (Ahmad et al., 2002), consistent with the oxidized flavin chromophore when the receptor is catalytically active. Bouly et al. (2007) show that green light (563 nm) can reverse th e effect of blue light on hypocotyl elongation in developing seedlings, consistent w ith previous reports of green light revers al of blue and red irradiation (Figure 8; Folta, 2004). Bouly et al. (2007) also show that this effect is cry dependent, since green-light-induced stem elongation is not present in cry double mu tants, supporting the green-blue reversibility model. The cry2 receptor ha s been shown to be light labile (Ahmad et al., 1998). Bouly et al. (2007) tested CRY2 accumulation in res ponse to pulses of blue light, and blue light followed by finite pulses of green light. The results show that blue light degradation of CRY2 can be reversed by a short, single pulse of green light, suggesti ng that the semi-reduced, active state is transient and subject to adjust ment by green wavelengths. These data provide evidence of a dichromatic modula tion of cryptochrome activity. Since cry2 has a profound effect on contro lling flowering time (Guo et al., 1998; Valverde et al., 2004), effects of green wavebands on flowering were observed in Arabidopsis
35 plants grown under short day (non-inductive) cond itions followed by transfer to blue, green, or simultaneous green and blue light conditions (Banerjee et al., 2007) Plants transferred to blue light flowered earlier than those remaining in white light. However, when plants were moved to blue and green light conditions, th e addition of green light negate d the blue light effect. Green light inhibition of flowering ag rees well with earlier studies where removal of green light enhanced flowering in marigold (Klein et al ., 1965), and addition of green light suppressed flowering in carnation and lettuce (Vince et al., 1964). In a mechanistic context, the levels of FT transcript were shown to only accumulate in blue light conditions, again illustrating the antagonistic effects of green light (Banerjee et al., 2007). It will be interesting to see how CONSTANS localization and stabili ty are affected by green waveba nds, as this central regulator is strongly dependent upon stabiliz ation imparted through cry2 (Moc kler et al., 1999; Valverde et al., 2004). The uncovering of a green-light-induced cr y sensing state presents many interesting questions. First, is the effect of green light simply that of negati ng blue light responses, or is the green light cry state involved in stimulating di screte responses/ interactions with other light response pathways? Second, how do green waveba nds affect other known cry responses? Is The Photosensor Class Complete? Clearly green wavebands play a role in regu lating plant growth and development, and at least a portion of the responses are dependent on cryptochromes. The extent that blue-green cryptochrome relations affect plant growth and development are of inte rest. However, clearly there is evidence of green light responses that are cry-independent. There are several logical candidates for such green light responses, as we ll as the possibility that green wavelengths are detected by a sensor which has yet to be disc overed. Zeiger and collea gues have proposed that zeaxanthin, a carotenoid required for the blue-gre en reversible stomatal regulation, toggles
36 between an active and inactive state depending on green and blue light levels (Frechilla et al. 2000). Other intuitive candidates provide a basi s for further investigation. An abundant flavoprotein was isolated from the membranes of Cucrubita pepo and Phycomyces (Hertel, 2005). The protein was later shown to have homol ogy with type-1 aquaporins (Lorenz et al., 2003). In vitro analyses indicated that the protein bi nds flavin and that the binding can be reversed with chemical or bl ue light treatment. Although the in vitro work by Lorenz et al. (2003) presents no evidence of light effects a bove 500 nm, the protein remains an interesting candidate as a green light receptor in vivo Such a protein would be a logical candidate for greenlight-induced stem elongation (Folta, 2004), since such robust short-term growth accelerations would likely require a ra pid change in turgor. CRY3 (or CRY-DASH) is anothe r flavoprotein with high local sequence similarity to cryptochrome photoreceptors and photolyases. Recen tly this protein has be en shown to act as a photolyase specific for single-st randed DNA (Selby and Sancar, 2006), suggesting that it would not likely be functioning as the green light photosensor for or phan responses. However, CRYDASH localizes to the chloroplast (Kleine et al., 2003), and the CRY1 protein from Vibrio cholerae binds RNA. Since at least one of the gr een light responses a ppears to require RNA metabolism (Dhingra et al., 2006), Cry3 genes are good candidates for roles in green light plastid transcript responses. In addition, cr y3 is the only photoreceptor that is up-regulated in green light (Dhingra et al., 2006; supplemental data). The existence of other sensors is implied by data in the literature. GCR1 (G-protein coupled receptor 1) in Arabidopsis thaliana has the strongest homology to the major photoreceptor of animal vision in terms of structural topology (C olucci et al., 2002). GCR1 has
37 been found to interact with the alpha subunit of the heterotrimeric G-prot ein in plants (Pandey and Assmann, 2004), and recently it has been shown to be required for blue-low-fluencemediated Lhcb induction in etiolated s eedlings (Warpeha et al., 2007) G-proteins have been pharmacologically (Neuhaus et al., 1993) and func tionally (Okamoto et al., 2001) linked to light responses. Since mutants in these components are r eadily available they c ould be tested in the suite of green light assays. Studi es of potential chromophores for plant light receptors identified all-trans retinal in tomato extracts (L orenzi et al., 1994), the same isomer later shown to be most active in fungal and microbial opsin s. Other evidence iden tifies retinal-binding proteins in plants and implicates retinoids in blue -light regulation of stomatal aperture (Paolicchi et al., 2005). Another potential contributor to green-light-indu ced responses is heliochrome, a hypothetical far-red/green light reversible heme-based receptor (Tanada, 1997), which has yet to be identified. Since green-light associat ed phenotypes are subtle and often require specialized equipment to visualize, large genetic screens ar e not practical for identif ying potential mutants. Yet, based on the literature, the loss of a non-cryptochrome green light sensor would be predicted to exhibit a lightdependent, hyper-photomorphogenic phenotype. Such mutants were isolated by Pepper et al (2001) in mutant screens conducte d under low fluence rate white and green light. Complete characteriza tion has not been reported. It is likely that components of the green light sensing system have been isolated from the many screens performed under red, blue, or white light. In support of this notion, many photomorphogenic si gnaling mutants have been well studied yet do not fit conveniently into cr y or phy pathways. These mutants represent an excellent starting point for further tests, now that several cry-inde pendent green light assays have been defined.
38 Conclusions Recent findings describing cry-dependent and cr y-independent responses add to evidence that green light provides developm entally-influential information to plants. For the most part, recent work agrees with previous findings i ndicating a negative role for green light in photomorphogenesis and strengthens the argumen t that these wavebands impart responses atypical for red or blue light. In this way, green light may be f unctioning in a manner similar to far-red light, serving as a cue for suboptimal conditions for photosynthetic activity. This possibility is supported biologically since green a nd far-red enriched environments are similar in nature. Many examples exist in biology in whic h multiple systems oppose each other in order to fine tune responses and conserve valuable resources. In this sens e, green wavebands may lead to conservation of valuable resources and extension into regions of more optimal light. Similar to the literature reviewed here, work in the followi ng chapters describes roles for green light which are atypical to other light-mediated responses.
39 Figure 2-1. Quantum ener gy distribution of full sunlight a nd under the shade of leaves (canopy shade). Light conditions were measured at noon in mid-April in Gainesville, FL (29.67 N), using a StellarNet spectrorad iometer (From Folta and Maruhnich, 2007).
40 Figure 2-2. Data reproduced from Wents green-depleti on experiments in 1957. Tomato seedlings were grown under white light or la vender filters (lower green light) and the dry mass of seedlings was measured after six days. The data show that at low fluence rates, red and blue light were more efficient than white light (red, blue, green) in promoting vegetative growth, as expected. Ho wever, at higher fluence rates the white light grown plants achieved a lower biomass than those grown in green-light-depleted conditions, even when light intensity was high. The author interpreted these findings as evidence for inhibition of growth by green light. (F igure adapted from F.Went, Experimental Control of Plant Growth, 1957).
41 Figure 2-3. The proposed photocyc le for plant cryptochromes. In darkness the cryptochrome flavin chromophore exists in the oxidi zed form (FAD), rendering the photoreceptor inactive, yet stable, thus facilitating it s accumulation to high levels. Stimulation by blue light drives reduction of FAD to FADH, the active signaling state (and green light absorbing state) of the receptor. Illumination with gr een light further reduces the chromophore to its fully-reduced form (FADH) that inactivates the receptor. Dark reversion returns the flavin from all redu ced forms to the oxidized, blue-absorbing state. (Figure adapted from Bouly et al., 2007).
42 CHAPTER 3 GREEN LIGHT EFFECTS ON EARLY DEVELPOPMENT Introduction The seedling undergoes a marked shift in developmental program as it transitions from growth in darkness to growth in light. Many environmental stimuli are integrated by plant systems and morphogenesis is altered to maximize su rvival and fitness. One of the most critical environmental cues is light, since light qualit y, quantity, and duration clearly affect seedling growth and development. In addition, light signals the seedling of time, place, and proximity to neighbors. Different wavebands drive suites of morphological, physiological, biochemical, and molecular changes that prepare the juvenile pl ant for optimal growth in its changing light environment. For young seedlings, the process of photomorphogenesis is typified by expansion of the cotyledons, opening of the apical hook, and induction of transcripts associated with lightinduced development. One of the most conspicuous changes is an alteration of stem growth rate. The rate is rapid in darkness, yet is quickly a nd robustly repressed when exposed to specific light qualities. Inhibition of hypocotyl growth rate depends on several separate photosensory systems that orchestrate downstream events wi th precision. Growth rate is st rongly suppressed by exposure to continuous UV, blue, red, or fa r-red light (Parks and Spaldin g, 1999, reviewed by Chen et al. 2004; Suesslin and Frohnmeyer 2003). In red light, st em growth is inhibited by phyA for the first 3 h, followed by the influence of phyB (Parks a nd Spalding, 1999). Blue light controls stem growth through phot1 for the first 30 min of i rradiation, followed by inhibition mediated by cry1, cry2, and phyA (Folta and Spalding, 2001a). This second phase is antagonized by phyB (Folta and Spalding, 2001b). It is not surprising that re d and blue wavelengths have profound effects in stem elongation since these regulate photosyntheti c rate in the developing seedling. The close
43 relationship between development and metabolis m aids adjustment of the newly emerged seedling to ambient conditions, through changes in gene expression that ultimately optimize body plan. While all other wavebands in the visible and flanking spectrum inhibit early stem elongation, monochromatic green light induces a robust, yet transi ent increase in stem growth rate (Folta, 2004). Green light i nduction of stem growth challenge s the currently-accepted model that etiolated seedlings exhibit the most rapid rate of elonga tion and that added light stimulates inhibition of hypocotyl elongation. Light added as green wavebands leads to a program more reminiscent of a skotomorphogenic growth. This acute green light response is not affected by mutation of known photoreceptors or co-irradiation with dim-red light, suggesting it is mediated by a novel sensory system (Folta, 2004). Anothe r response linking green light to programs conflicting with red and blue growth is chlo roplast transcript accumulation (Dhingra et al., 2006). This work clearly demonstrates that green light induces a decreas e in specific plastid transcripts which opposes their red and blue induction (Dhingra et al., 2006). In these experiments, a short, pulse of green light, like that in the above studies was found to decrease certain chloroplast tr anscripts (such as psaA and psbD ) that are central parts of photosystems I and II. This effect of green light agrees with periodic reports during the last 50 years that proposed a negative role for green light during de-etiolation (see Chapter 2 for references). Regulation of chloroplast transcript accumula tion in this manner could have a selective advantage for resource conservation, since green light is much less effective at stimulating photosynthesis when compared to red and blue light and is often enriched in environments with low light or photosynthetically acti ve radiation (PAR). It follows that in this and in stem
44 elongation, green light (or a high green to blue or red ratio) may signal a suboptimal light environment. Monochromatic light treatments have allowe d characterization of specific light sensing systems and their contributions to plant growth and development. Combinatorial light treatments are less prevalent in the literature. However, coaction between blue and red sensing systems has been examined (Fuglevand et al., 1996; Ahmad and Cashmore, 1997; Guo et al., 1998; Neff and Chory, 1998; Guo et al., 2001) and tests of red, blue and/or green light supplementation in the growth of specific crop plants has been pres ented (Kim et al., 2004, 2004). Similarly, recent work by Bouly et al. examines the effects of combinatorial light conditions on Arabidopsis seedlings using broadband white light supplemen ted with green LED sources (2007). Since green wavebands influence early events in stem growth (within 15 min) and also influence longterm growth (days), it is important to test th e interaction and crosstal k between light sensing systems. Given that monochromatic gr een light has been shown to induce effects that oppose normal photomorphogenic growth, experiments in th is chapter were devo ted to assessing green light effects in the presence of red and blue light during early s eedling growth. Based on influence of monochromatic green light in st em and chloroplast transcript regulation, our hypothesis was that green light would negative ly affect photomorphogenic progression in early seedling development. Seedlings were grown under narrow-bandwidth, red, blue, and green light-emitting diodes (LEDs) that allowed precise control of light quality and quantity, thus decreasing the extraneous wavebands present in broadband light sources. Therefore, the phenotypes observed can be more tightly co rrelated with each of the wavebands.
45 Results Hypocotyl Elongation Initial experiments demonstrated that suppl emental green light (G) added to a background of red and blue light (RB) increased hypocotyl elongation, increased lateral root number, and slightly delayed true leaf deve lopment relative to RB controls (hypocotyl data: Figure 3-1; P < 0.0001). The first experiments utilized condition s similar to those reported by Folta (2004), where administration of low-flue nce rate light demonstrated a role for monochromatic green light in stem elongation. RB+G tr eatments had a higher fluence rate (an additional 1mol m-2 s-1) relative to RB, yet generated long er hypocotyls (a phenotype associat ed with lower-light or dark conditions). Therefore, it was unlikely that the re sulting phenotype was due to increased light in RB+G treatments. To test this possibility, plan ts were grown under either supplemental green (G), red (R), or blue (B) light added to a background of RB (Fi gure 3-1). For these experiments RB was set to R= 2 mol m-2 s-1and B= 2 mol m-2 s-1 and +G/+B/+R added at 1 mol m-2 s-1. Hence, the total photosynthetic photon flux (PPF) was 4 mol m-2 s-1 for RB and 5 mol m-2 s-1 for RB+G, RB+B, and RB+R treatments. Plants were measured after 10 d of treatment. Although hypocotyl growth is typically measured earlier in development (2-6 d) the 10-d point allowed for appraisal of the low-fluence phe notype at its most significant phase. After 10 d of treatment, plants grown under RB+B had shorter hypocotyls than those grown in RB, and hypocotyls of plants grown in RB+R conditions were the same or shorter than those from RB treatments. These results indicate that hypocot yl elongation observed in plan ts grown under RB+G was caused by green wavebands and not increased light when compared to those under RB. These experiments also indicated that the increased lateral root phenotype observed in RB+G was due to increased light, or photosynthetically active radiation (PAR), rather than to added green light specifically, since this phenotype was also obs erved under RB+B and RB+R treatments.
46 Green-Light-Induced Hypocotyl Elongation is Dose-Dependent To determine the photophysiological paramete rs of the green light response, fluence rate/response experiments were carried out wher e increasing amounts of green light were added to a background of RB that equaled the 4 mol m-2 s-1 delivered for experiments shown in Figure 3-1. Increasing amounts of green light were administered as 0, 1, 10, and 20 mol m-2 s-1 (Figure 3-2). Data in Figure 3-2 demons trate that although small amo unts of green light (1mol m-2 s-1) added to a background of RB promote hypocotyl elongation, quantities of 10 and 20 mol m-2 s-1 inhibit elongation. As was the case for monochrom atic green, green light added in quantities which exceeded a given fluence ra te (between 1 and 10 mol m-2 s-1) inhibited hypocotyl elongation, most likely due to gr een light stimulation of the phytochrome and cryptochrome photosystems. Other Observations In addition to effects on hypocotyl elongation, leaf development in seedlings grown under supplemental green light appeared delayed. Leaf pa rameters observed were: petiole length of the first leaf, and anthocyanin conten t and chlorophyll content. Petiole length of the first leaf was measured under all light conditions (RB, RB+G RB+R, and RB+B) (data not shown). In RB+R and RB+B conditions, the petiole lengths of the fi rst leaves were predictable and consistent between experiments. However, RB+G effects were variable between experimental replicates. In some instances, significant differences were obser ved, but in others, variab ility was evident that could not be attributed to the e xperimental treatment. Therefore, no consistent effect of green light on petiole length was apparent in the pres ent studies. Although leaf area was not measured for these treatments, the leaf area of seedlings grown under supplemental green light had visually smaller overall leaf area than those grown unde r RB+B and RB+R treatments. The largest leaves
47 formed under RB+B conditions, followed by smaller ones from the RB+R treatment, and the smallest leaves in RB and RB+G environments. Anthocyanin and Chlorophyll Levels Since supplemental green light decreases anth ocyanin levels in mature plants and in seedlings (mature plants: Chapter 4 of this wo rk, seedlings: Bouly et al., 2007), anthocyanin levels were examined in these experimental conditions. Chlorophyll content was also examined since leaves under +G treatments appeared lighter green than t hose from +R and +B conditions. Both metrics were quantified af ter 10 days treatment under conditions that stimulated stem elongation. In Figure 3-3, data from these experi ments is expressed as absorbance at a given wavelength (530 nm for anthocyanins and 657 nm for chlorophyll) per gram fresh weight. No discernable trends emerged from anthocyanin or chlorophyll assays at this developmental time point and at these fluence rates. Since green light inhibits anthocyanin accumulati on in mature plants (Chapter 4 this work) and seedlings (Bouly et al., 2007), and no discernable differences were detected in the data presented in Figure 3-3, it was im portant to examine the effect of higher fluence rates of green light. The rationale was that the quantum efficiency of cry absorption decreases as the wavelength becomes red-shifted, so higher fluence rates may be required to induce cry reversal. While anthocyanin levels were similar under RB+G (G= 10 mol m-2 s-1) treatments relative to RB+G (1 mol m-2 s-1) and RB, anthocyanin levels we re greater under RB+G (20 mol m-2 s-1) treatments. Photoreceptor Mutants Green-light-induced hypocotyl elongation was examined in known photoreceptor mutants. The cry1 cry2 and phyA phyB mutants were grown under the es tablished conditions where green light effects were conspicuous. Figure 3-5 de picts the results of these experiments.
48 Under all treatments, the photoreceptor mutant s exhibited longer hypocotyls than did wild type seedlings. This was expected, since thes e mutants lack the red light and blue light photoreceptors that inhibit hypocotyl elongation. Th erefore, RB treatments were set to a value of 1 so that growth patterns could be better compared to wild type trends. First, cry1 cry2 double mutants were examined. There was no significan t difference in hypocotyl length detected for cry1 cry2 mutants after 10 d under any of the conditions tested. These data are consistent with cryptochromes mediating green-lig ht-induced elongation, similar to experiments by Bouly et al. (2007). In addition to testing the major sensors i nvolved in blue-induced inhibition of hypocotyl elongation, effects of the major contributors to red-induced inhibiti on (phyA and phyB) were examined (Figure 3-5). These mutants responded as wild type to green-l ight-induced hypocotyl elongation. These data indicate th at neither phyA nor phyB are re quired for green-light-induced stem elongation. Discussion Green light antagonizes normal photomorphogenic gr owth when delivered to an etiolated seedling in the presence of red and blue light. Si milar to monochromatic green light studies, low fluence rates of green light added to a red and blue background promoted hypocotyl elongation, whereas higher fluence rates inhi bited elongation. Previously iden tified green light effects in seedling development are subtle and detected only with minute-to-m inute high-resolution imaging equipment (Folta, 2004), or by analysis of specific gene expression (Dhingra et al., 2006). However, in this report we present more prominent, visually clear evidence that green light opposes the normal progression of light-med iated development. These findings expand and further support previously publis hed data that seedlings grow taller when supplemented with green light (Bouly et al., 2007; Folta, 2004). Coll ectively, these findings constitute an exception
49 to the accepted dogma that hypocotyl elongation rate is proportionately limited by higher fluence rates of light. Although green light has been found to decrease anthocyanin levels in seedlings (Bouly et al., 2007) and in mature plants (Chapter 4 of th is work), green-light-i nduced down-regulation of anthocyanins was not observed under these c onditions. These differences in green light responses may be due to variati ons in developmental stages. Howe ver, work done by Bouly et al. (2007) demonstrated that green light down-regu lated anthocyanin accumulation in early seedling development, which is consistent with mature pl ant studies (Chapter 4). This suggests that the lack of green-light-induced down-re gulation in these experiments is more likely due to fluence rates insufficient to stimulate an thocyanin production in the first place. Experiments in Bouly et al. (2007) and Chapter 4 of this work observed th ese effects under greater fluence rates compared to work described within this chapter. Here, supplemental green (blue and red) was added as 1 mol m-2 s-1 to a background of 4 total mol m-2 s-1 RB. These treatments represent very low fluence rates of light. Lack of green-light-i nduced down-regulation may be the result of insufficient up-regulatio n of anthocyanins. However, green light added at a level of 20 mol m-2 s-1 to a background of 4 mol m-2 s-1 RB induced an increase in anthocyanin accumula tion. As in stem elongation, this quantity of green light likely stimulates crys and phys, both of which are known to stimulate anthocyanin accumulation. Whether or not this increase in an thocyanin accumulation in plants grown under 20 mol m-2 s-1 green light (G) is directly linked to green wavebands can be addressed by examining anthocyanin levels under similar fl uence rates of supplemental red and blue. Interestingly, Bouly et al. (2007) found th at green light down-re gulated anthocyanin accumulation under conditions supplemented with green light (20). However, experiments by
50 Bouly et al. (2007) also utili zed greater background fluence ra tes than used in the present experiments. This may explain differences in observed phenotypes. Unde r conditions described within this chapter, increased anthocyanin accumulation under RBG (20) may be the result of increased leaf development when comparing RBG (20) to all other treatments. RBG (20) treatments also accumulated more chlorophyll than RB, RBG (1), and RBG (10). This may also be related to increased leaf development in RBG (20) when compared to other treatments. After examination of green light effects in early development, photoreceptor mutants were tested under the conditions where green-depende nt effects were obser ved. Although green-lightinduced elongation is opposite to red and blue inhibition, crys and phys absorb green light and crys have been implicated in green-light-induced stem elongation in earl y development (Bouly et al., 2007). Wild type promotion of hypocotyl elongation by supplemental green light was not observed in cry1 cry2 mutants indicating that crys are required for green-light-mediated stem elongation. An argument can also be made that cry mutants are maximally elongated and cannot further increase their length under supplem ental green light because mutants under all conditions, even +R (where phy inhibition is pr esent), exhibited equally elongated hypocotyls. However, red-induced inhibition is not robust at these fluence rates. In addition, other mutants (such as phyA phyB mutants) were able to expand beyond th is length (Figure 35), indicating that crys are physically able to e xpand and the lack of increased elongation under supplemental green light results from loss of crymediated, green-light-induced res ponses. To further clarify this issue, cry double mutants can be observed under increa sed RB backgrounds in order to stimulate phy-mediated inhibition of elongation.
51 In addition, phy mutants were examined. The phyA phyB mutants demonstrated wild type trends, i.e. supplemental green light induced hypoc otyl elongation. These data imply that neither phyA nor phyB are required for this green light response. Experiments in this chapter provide evidence of a role for green light in early stem elongation which opposes forward photomorphogeni c progression, and the effects of red and blue light. These experiments reinforce previ ous work, which showed monochromatic greenlight-induction of stem elongation (Folta, 2004). This work also supports recent findings that crys are required for green-light-induced stem elongation during seedling development (Bouly et al., 2007). Materials and Methods Plant Materials Arabidopsis thaliana genotypes tested are identical to those previously assessed for responses to blue (Folta and Spalding, 2001, 2001) and green light (Folta, 2004) light; cry1-304 cry2-1 mutants (in the Col-0 background), phyA201 phyB-5 mutant (in the L er background), and Col-0. Wild type references within the text refer to Col-0 ecotypes. Light Sources and Treatments Actinic light treatments were generated using narrow-bandwidth LED light supplied by American Bright LEDs driven by custom electroni cs as described (Folta et al., 2005). Fluence rate was further attenuated with neutral density filters (layers of M209 and/or M-211, Cinemills Corporation, Burbank, CA) when needed. The emi ssion spectrum of all light sources is viewable on-line at www.arabidopsisthaliana.com/lightsources. Light composition was assessed using an Apogee spectroradiometer and Spectra Whiz software.
52 Hypocotyl Elongation Assays Arabidopsis seeds were sterilized using the following protocol: seeds were immersed in 0.01% Tween 20, shaken for 15 minutes, at which point liquid was evacuated and 50% bleach added plus shaking for 10 minutes, rinsed several times (2-3) with sterile diH2O, quickly washed with 70% ethanol, and then rinsed 8-9 times with sterile diH2O and dried in the laminar flow hood. Once dry, seeds were sprinkled on media c ontaining 10 ml/ L macronutrients for MS media, 500 l/L iron for MS media, 0.50 g/L MES, 1% sucrose, and 1% Phytoagar, with a pH of 5.8. The plates were covered in foil, stratified for 48-72 h at 4C, and then the seeds were treated with 5 min to 1 h of fluore scent white light (~16 mol m-2 s-1) depending on genotype requirements (i.e. Col 5 min, cry1 cry2 mutants 40 min, phyA phyB mutants requires longer treatments--1 h in order to effectively stimulate germination). Seedlings were grown in absolute darkness at 23C for approximately 40 h, and then germinated seed lings of similar size were transferred to square Petri pl ates containing previously desc ribed media under dim white light (<1 mol m-2 s-1). Exposure to white light prior to transfer to expe rimental conditions did not affect the experimental outcome (not shown). Uniform etiolated seedlings were distributed across the plate. The seedlings were imaged w ith an 8.0 megapixel digital camera with an appropriate size standard (time 0), and then were moved to experimental light conditions. Seedlings were imaged at various intervals within a 10 day period. For measurement of stem elongation, plates we re scanned after 10 days of treatment and images were imported into ImageTool 3.0 software where they were measur ed relative to a size standard. The length of hypocot yls in pixels was determined using ImageTool software on enlarged images with 0.01 mm re solution. Pixels were converte d to linear measurement using size standards. Seedlings height was graphed in Excel.
53 Anthocyanin and Chlorophyll Levels Aerial portions of seedlings were harveste d after 10 d of treatment weighed and either moved to -80C for storage or soaked directly in 100% methanol: 1% HCl, vortexed, and placed at 4C in the dark (wrapped in foil) overni ght. The next morning samples were vortexed, centrifuged quickly, and absorbance was measured at 530 nm (for anthocya nin) and 657 nm (for chlorophyll). Quantities were then expressed as the total absorb ance at a given wavelength per fresh weight of the sample (in grams).
54 Figure 3-1. Supplemental greenlight-induced hypocotyl elonga tion is specific to green wavebands. 10-d-old Arabidopsis plants were grown under RB conditions (R= 2 mol m-2 s-1, B= 2 mol m-2 s-1) with supplemental G, R, or B= 1 mol m-2 s-1 (Yaxis). Hypocotyls were measured as de scribed in Materials and Methods. The RB treatment alone was set to 1 and the experime ntal data are shown as length relative to RB. Asterisk indicates statistical differe nce between RB and RBG treatments based on students t-test (P < 0.0001). Each column represents at least 20 seedlings in at least 2 experimental replicates. Error ba rs show standard error of the mean.
55 Figure 3-2. Fluence rate/response experiment. 10-d-old Arabidopsis plants were grown under RB conditions (R= 2 mol m-2 s-1, B= 2 mol m-2 s-1) and supplemented with G at 0, 1, 10, and 20 mol m-2 s-1 (Y-axis). Hypocotyls were measured and data are expressed relative to hypoc otyl lengths under RB (0G). Da ta represent at least 20 seedlings and at least 2 expe rimental replicates per treatment. Error bars represent standard error of the mean.
56 Figure 3-3. Anthocyanin and chlorophyll levels for 10-d-old Col seedlings grown under RB with +G, +B, and +R as indicated (Xaxis). A) Anthocyanin levels measured as the absorbance of the sample at 530 nm per gr am fresh weight (Y-axis). B) Chlorophyll levels measured as the absorbance of the sample at 657 nm per gram fresh weight (Yaxis). At least 20 seedlings and 2 experime ntal replicates were quantified for each treatment. Error bars represent the standard error of the mean. A B
57 Figure 3-4. Anthocyanin and chlorophyll leve ls for 10-d-old Col seedlings grown under increasing fluence rates of green light. For these experiments, RB was given with R= 2 moles m-2 s-1 B= 2 moles m-2 s-1 with increasing amounts of green light at the following fluence rates: 0, 1, 10, and 20 moles m-2 s-1 (X-axis). A) Anthocyanin levels as measured by the absorbance of the sample at 530 nm/fresh weight (g) (Yaxis). B) Chlorophyll levels as measur ed by the absorbance at 657 nm of the sample/fresh weight (g) (Y-axis). At least 20 seedlings and 2 experimental replicates were observed for each treatment. Error bars represent the standard error of the mean. A B
58 Figure 3-5. Hypocotyl elongation experiments for photoreceptor mutants (Wild type [Col], in purple, cry1 cry2 mutants in blue, phyA phyB mutants in green). Average hypocotyl lengths were measured after 10-d with RB and +G, +B, +R treatments for all genotypes. Quantities are expressed relativ e to RB with average hypocotyl lengths under RB set to 1 for all genotypes. At least 20 seedlings and 2 experimental replicates were performed for these experi ments. Error bars represent the standard error of the mean.
59 CHAPTER 4 GREEN LIGHT EFFECTS ON MATURE PLANTS Introduction Plants are anchored organisms, therefore thei r capacity to compete and survive relies on the extent to which they can sense and res pond to even minute cha nges in their ambient environment. Incident irradiation provides an important package of e nvironmental information. Light quantity, quality, and dur ation all have meaningful effects on plant growth and development (Chen et al., 2004; Spalding and Folt a, 2005). One example of a plant response to changes in the light environment is the shade-av oidance syndrome, a genetic program that alters plant form and gene expression to acclimate to a light-limited environm ent. Induction of this response is regulated by red: far-red ratios. Low ratios of red to far-red light reflect shading and high plant density, as far-red light is readily transmitted through plant tissues in the canopy while red and blue wavelengths are mainly absorbed by overhanging foliage (Smith and Whitelam, 1997; Ballare, 1999; Kim et al., 2005; Vandenbussche et al., 2005). Other light environments that initiate shade-avoidance re sponses are low levels of blue light or photosynthetically active radiation (PAR, 400-700 nm). Like far-red light, gr een light can also pass through plant tissue and is present in higher amounts than red and blue in the understory of a canopy (Klein, 1992). While little is known about green light sensing and signal transduction, ex periments suggest that green wavebands provide important informati on. Supplemental green light seems to oppose deetiolation responses that are in itiated by red and blue light (C hapter 2, Klein 1992; Folta 2005; Went 1957; Klein et al., 1965; Kim et al., 2004a; Kim et al., 2 004b; Frechilla et al. 2000, Talbott et al., 2003; Folta, 2004, Br udler et al., 2003). In early seedling development, specific exam ples of this apparent opposition between green light and red or blue light have been described. For example, green light promotes
60 hypocotyl elongation during early se edling development and this rate of elongation surpasses dark-growth rates. In contrast, growth rates are inhibited by all other light qualities tested (red, blue, far-red, UV-B) (Folta 2004, reviewed Chen et al. 2004; Suesslin and Frohnmeyer 2003). In addition, a brief pulse of dim green light causes a decrease in the accumulation of specific plastid mRNAs (Dhingra et al., 2006). These transcripts encode proteins required for photosynthesis and they typically increase in abundance after red or blue light treatments (Dhingra et al., 2006). In addition green light was found to induce hypocotyl elongation in combinator ial light treatments (Chapter 3). These are three examples in which green wavebands play an antagonistic role to normal photomorphogenic development. Green-light -enriched environments are usually also enriched for far-red light. Therefore, green li ght may signal a suboptimal light environment much as do far-red wavelengths, and thus induc e phenotypes similar to those of the far-redinduced shade-avoidance syndrome. Another goal of this work was to test candida te genes for roles in green light pathways. Recent work by Bouly et al. (2007) and Chapter 3 of this work i ndicate that cryptochromes are involved in green light sensing as it pertains to early stem elongation in combinatorial light conditions. Bouly et al. (2007) demonstrate that when the cr yptochrome flavin chromophore absorbs blue light it is converted to a green-light-absorbing semi quinone. Green light then reverts the active light sensor state to an inactive state. Bouly et al. ( 2007) suggest that the cryptochrome receptor may toggle between an inactive, blue-li ght-absorbing, and active, green-light-absorbing, form much like phytochrome receptors. While B ouly et al. (2007) demonstrate cry-dependence; other studies show cry-independence (F olta 2004) of green light responses. Experiments described in this chapter test the effects of green light in the presence of red and blue wavelengths. To isolate the roles of gr een light, far-red light has been excluded from
61 experimental conditions. Custom, adjustable LED (light-emitting diode) lighting systems were built so that specific quantities and qualities of light could be administered. Experiments were conducted to test the hypothesis that green light, represented by wavebands generally considered to be photomorphogenically inert, will induce adjustments in morphology characteristic of shade-avoidance symptoms induced by far-red enriched environments. Results Plants were grown for 15 d under RB conditions (~65 mol m-2 s-1 R light, ~15 mol m-2 s-1 B light) or supplemented with green light (RGB ; same as RB treatment with ~15-20 mol m-2 s-1 G light). Representative plants from each tr eatment are shown in Figure 4-1. Plants grown under RB conditions displayed normal photomorphogenic growth, i.e. open and expanded leaves, leaves oriented relatively parallel to soil, and shorter petioles, compared to plants grown under RGB. Phenotypes observed under RGB mirrored those of plants subjected to low red, high far-red light environments. Plants showed charac teristics associated with the shade-avoidance response despite having been grown under higher fl uence rates of light (Figure 4-1). Plants grown under supplemental green had smaller leaf blades, longer peti oles, and displayed a hyponastic response when compared to plants grown under RB. Rosettes from over 19 individual 15-d-old plan ts were dissected from two separate experiments. An entire leaf whorl from a represen tative wild-type plant is presented in Figure 42A and exhibits a conspicuous, shade-avoidan ce habit under RGB. A se ries of morphological metrics were measured including leaf length, leaf bl ade area, leaf blade length, leaf width, petiole length, and leaf inclination angle. All indicated symptoms consis tent with the shade-avoidance syndrome. The most diagnostic measurements were petiole length as a f unction of total leaf length (Figure 4-2B) and leaf blade area (Figure 4-2C). These were measured for one of the leaves from the second pair of true leaves (ref erred to as the second leaf from here on). These
62 experiments demonstrate that supplemental green light induces elongation of petioles. The data are presented as petiole length rela tive to total leaf length (Figure 4-2B), since the latter varied slightly. The percent of the tota l length occupied by pe tiole is a much more dependable indicator of the phenomenon among all genotypes studied. The addition of green wavebands leads to increased petiole elongation at the expense of the leaf blade. The petiole represented 42% of the total leaf length under RB, yet comprised 56% of the RGB leaf (Figure 4-2A and 4-2B). Similar results were observed for Col-0 and L er ecotypes although phenotypes were more prominent in Col-0 ecotypes (L er not shown). Leaf blade area clearly was affected by additi onal green light. The re sults indicate that addition of green light to RB re duced leaf area by 64% when comp ared to control plants grown under RB treatments alone (Figure 4-2A and 4-2B). Th is finding is contrary to the accepted rule that irradiation with a greater number of photosynt hetically-active photons causes increased leaf expansion. Similarly, clear hyponastic leaf orient ations were observed in the upright growth habit of leaves, which is consistent with lowe r-light-induced phenotypes. A capacity to reach over neighbors may provide selectiv e advantage to plants and enrich fitness. Under RB the first and second pairs of rosette leaves were positio ned parallel to the soil with only minor upward deviation. Under RGB the elongated petioles angl e sharply upright, reaching above the rosette plane (Figure 4-1). The results indicate that the simple addition of narrow-bandwidth green light promotes the shade avoidance response. Green-light-induced shade-avoidance responses in mature plants do not likely result from activation of known photosensors because it is observed under constant blue and red light conditions that excite, if not saturate, known photosensory pathways. However, there are several reasons to test the green light response in photomorphogenic mutant s. First, blue-light sensing
63 cryptochromes have been shown to absorb green light and have been found to be required in stem elongation in early seedling development (B ouly et al., 2007; Chapter 3) and flowering (Banerjee et al. 2007). We also tested phytochrome mutants for their ability to respond to green wavebands. Phytochromes absorb in the green region of the spectrum and contribute to shadeavoidance responses. However, reported phytochrom e effects are induced by enriched far-red, or low ratios of red to far-red, and there is little to no far-red und er these conditions (<.0001). Also, red light was enriched under our conditions, whic h would have stimulated phytochromes to their active PFR form. This in turn would have promoted positive photomorphogenic growth and a full-sun growth habit. Therefore, if phys are directing shade-avoidan ce syndrome through green light it would require participati on of a novel mechanism. Another r eason to test these mutants is that if the influences of redand blue-light-sensing system s that advance photomorphogenic development are removed genetically, the genetic mechanism that underlies the inhibitory green light system may become even more apparent. Responses to experimental conditions for Figur e 4-1 and 4-2 were also tested using red and blue photoreceptor mutants: phyA phyB cry1 cry2 and phot1 phot2 mutants. Except for phot1 phot2 mutants, the leaf metrics described in Figure 4-2 were measured for the second leaf of each of these mutants. Lack of phyA and phyB or cry1 and cry2 receptors consistently and significantly resulted in amplification of the e ffects of green light. The addition of green light resulted in increased petiole length in the sec ond leaf under both RB and RGB conditions for the mutants. Therefore, differences in petiole length s were not as prominent as in wild-type plants. This was expected, since these mutants are missi ng the genetic elements that inhibit elongation, therefore are much more elongate d than wild type plants under RB conditions. These organs have less potential to elongate so the differences between RB and RGB would be of a lesser
64 magnitude. However, general trends were consistent with responses of wild type plants, since the addition of green light enhanced growth. For each of these mutants, the addition of green light also decreased leaf blade expansion relative to the RB-grown plants (F igure 4-3A and 4-3C). With supplemental green light, leaves of phyA phyB mutants only expanded to 54% of those from RB-grown plants. In cry1 cry2 mutants, this was more pr onounced, since added green light limited expansion to only 38% of that observed for leaves under RB conditions. These values can be compared to those of wild-type plants, wh ere RGB conditions limited leaf expansion to 64% of RB alone. In addition to i nhibiting petiole elongation, ac tivation of cryptochromes and phytochromes has been shown to increase leaf expansion (Cerdan and Chory, 2003; Kozuka et al., 2005). The results presented here are consis tent with the hypothesi s that a green light transduction system opposes mechanisms of RB-medi ated leaf expansion. The differences in leaf expansion were more apparent than those in pe tiole elongation for thes e mutants (Figure 4-3A compared to 4-2A). In general, when phy and cr y receptors were excluded, the effects of green light persisted but were less conspicuous. These genetic stud ies indicate that the major phytochromes and cryptochromes are not requir ed for green-light-induced shade-avoidance responses. It became immediately obvious that the phot1 phot2 mutants were not useful to these analyses because the phot1 and phot2 photosensors are required for robust light-mediated leaf expansion (Sakamoto and Briggs, 2002). Greenlight-induced opposition of expansion cannot be accurately measured in the absence of vigor ous phot-mediated expansion. Leaves for these mutants were considerably reduced and shrivele d so accurate measurements were unattainable. However, although not possible to quantify, shade avoidance trends were visibly evident in phot1 phot2 plants grown under supplemental green light (data not shown).
65 Effects of increasing fluence rates of green li ght to a fixed background of red and blue light (RB conditions as in Figure 4-1) was examine d. Since the response was robust with the addition of only 15mol m-2 s-1, the threshold for the response was evaluated for 0.5, 4.5 and 15 mol m-2 s-1 of supplemental green light. Results in Figure 44 indicate that even a small quantity of green light photons (s-1) can significantly influence the shape and position of leaves. Figure 4-4A shows representative plants gr own under 0.5, 4.5 and 15 mol m-2 s-1 of added green light. Very little difference was observed in re sponse to addition of 0.5 mol m-2 s-1 green light. An obvious change in leaf inclination can be induced with a thresh old between 0.5 and 4.5 mol m-2 s-1, and is more pronounced at the higher green fluence ra te. Petioles of plants grown under 4.5 mol m-2 s-1 supplemental green light were 10% l onger than those grown under 0.5 mol m-2 s-1 added green light, and petioles from plants grown under 15 mol m-2 s-1 were 18% longer than those grown under 0.5 mol m-2 s-1 green light (Figure 4-4B). Meas urements taken from the second leaf of individual plants show that the addition of 4.5 mol m-2 s-1 green light to a background of red and blue induces a significant decrease in leaf area. These data (Figure 4-4C) also demonstrate that higher fluence rates of green light did not lead to a significant further cha nge in leaf area. The quantitative data presented in Figure 4-4 are slightly different from those presented in Figure 4-1, despite th e common ecotype and experimental light treatment. This discrepancy can be reconciled by age and pretreatment. Plants used in later experiments were permitted a l onger time to germinate and grow under white light (96 h vs. 1 week) before moved to experimental conditions. The longer germination duration was chosen because the traits of inte rest were more robust and consistent when plants were permitted to grow under a complete spectrum prior to transfer to experimental conditions.
66 The observed fluence-rate/response relationship indicates that the green light signal affects the growth of plant organs in a dose-dependent manner and these results are consistent with green-light-induced phenotypes observed in Figures 4-1 and 4-2. The low threshold is evidence that a sensitive photosensor modulates the response to supplemental green light. These experiments were repeated and observed over a 3 week period. At the end of this period, plants grown under RGB (G= 15 mol m-2 s-1) were larger than plants grown under RB or RGB (G= 4.5 mol m-2 s-1) conditions (observation). Thos e receiving RB and RBG (G= 4.5 mol m-2 s-1) treatments were similar in size. Plan ts grown under RBG (4.5) showed leaf hyponasty similar to 2-week-old Arabidopsis pl ants grown with G at either 4.5 mol m-2 s-1 or 15 mol m-2 s-1 (Figure 4-1), whereas plants grown under RBG (15) did not. The size of the leaf blades of plants grown under RBG (15) tr eatments may have negatively impacted leaf inclination, since a substantial part of the blade is far from the st em. This increase in resistance distance could have led to lower leaf inclination. Interestingly, after thr ee weeks of treatment, approximately 45% of plants grown under RB and RBG (4.5) treatments were flowering while this was evident for only 8% of plants grown in RBG (15) treatments. Data are consistent with previous observations (Kim, Hye on-Hye, K. Folta; not shown). Another interesting observation was a visi ble difference in anthocyanin accumulation between 3-week-old plants grow n under increasing fluence rates of green wavelengths (Figure 45). Added green light (increasing from left to right in Figure 4-5) resulted in decreased anthocyanin accumulation. Differences were most ev ident at the base of petioles. This region was darkest purple in plants grow n under RB treatments, with slight ly less pigment detected in plants from RGB (G= 4.5 mol m-2 s-1) treatments, and anthocyanins were barely visible in plants grown under RGB (G= 15 mol m-2 s-1) conditions.
67 Recent work by Bouly et al. (2007) demonstrat ed that green-light-induced regulation of anthocyanin accumulation was mediated by cryptochro mes. To test this finding with our lighting system cry1 cry2 double mutants were grown under RB and RBG (15) conditions and anthocyanin levels were compar ed to those of wild type plants grown under RB and RBG (15) conditions. For these treatments RB and RBG were administered with R= 45-55 mol m-2 s-1, B=13-15 mol m-2 s-1, and G=14-15 mol m-2 s-1 respectively. The quantit ies of red light are slightly less in these experiments than in previous experiments (Fig 4-1 and 4-2), because this is the maximum quantity attainable for these LED ch ambers. However, these quantities of red light are given at levels that fully establish a high PFR to PR equilibrium and saturate photosynthesis, as shown in comparable studies by Dr. HyeonHye Kim (personal communication). Wild type Arabidopsis plants grown under RB and RBG (15) treatments showed similar trends as in the previous experiments (Fig 4-1 and Fig 4-2). The cry mutants maintained wild type responses to added green light, i.e. down-regula tion of anthocyanin accumulation. To expand this work to horticu ltural crops, diploid strawberry, Fragaria vesca was tested under RB and RGB conditions. The disadvantage of studying strawberry is that only a small number of plants can be subj ected to a given experimental condition at the same time. Nonetheless, preliminary results indicate that small amounts of supplemented green light, as in Arabidopsis plants, influence pl ant growth and development. Mo reover, green light effects are consistent with low-light phenotypes. Strawberry plants grown under RGB were co mpared to those under RB, with R= 45-55 mol m-2 s-1, B=13-15 mol m-2 s-1, and G=14-15 mol m-2 s-1 respectively. Plants were monitored over a nine week period and the follo wing parameters were observed: leaf number, number of fully-differentiated leaves (leaves w ith 3 leaflets/ petiole), number and length of
68 runners, number of plants flower ing at nine weeks, color of leaves, anthocyanin accumulation, petiole length and leaf area. Data after nine weeks of treatment i ndicated that mean responses of RBG-treated plants (Figure 4-7) were reduced for every parameter examined. When compared to Arabidopsis, Fragaria vesca responses showed some similar trends. In both species, plants appeared to be more vi gorous under RB conditions, whereas plants receiving RBG treatment appeared less healthy, despite th e increased photon flux. Pl ants under the latter conditions also had characteristics similar to lower-light phenotypes. Plants grown under RB conditions had more runners per plant (2 or more runners were present in 10 out of 13 RB-grown plants, 4 out of 7 RBG-grown plants) and more advanced leaf morphology (i.e. more fully differentiated leaves (3 leaflets / petiole) (Figure 4-7). Petiole lengths were the same under both light treatments, which differs from the incr ease in petiole elonga tion seen under RBG for Arabidopsis plants. No clear flow ering effects were observed. Diffe rences in anthocyanin levels were visible in petioles. Petiole s collected from 4 plants from each treatment demonstrated that supplemental green light decreased anthocyanin levels, much as seen in Arabidopsis plants. For anthocyanin levels, the absorbance of each sample was read at 530 nm (A530 nm) and reported relative to fresh weight (grams). A530 nm/g levels were 106.9 (+/28.6) for RB treatments, and 46.1 (+/10.27) for RBG (15) treatments. Leaf area of the largest leaf from each plant was measured and averages were the same between treatments (data not shown). Thus far, data indicate that supplemental green light affects pl ant growth and development in multiple species. Discussion Experiments described in this chapter dem onstrate that green wavebands, like far-red, induce shade-avoidance phenotypes. Here, added green light caused an increase in petiole length, decrease in leaf area, and in crease in leaf hyponasty in a dos e-dependent manner. In addition, supplemental green light decreased anthocyanin accumulation when compared to RB. These data
69 support the hypothesis that a gr een-light-sensing system te mpers physiology, growth, and development driven by red and blue light. Although green-light-induced stem elongation in early seedling development was found to be cry-dependent (Chapter 3; Bouly et al., 2007), a ll photoreceptor mutant s tested in these experiments demonstrated wild-type trends. Results indicated that there may be several sensory systems which initiate green light signals. In addition, these experiments demonstrate si milarities and differences between far-redand green-light-induced respons es. Far-redand green-light-e nriched environments induce petiole elongation and leaf hyponasty. However, far-red also induces early flowering, whereas green light delays it (Kim, H-H and K. Folta; unpublished obs., observation in this chapter). Relationships between far-red and green light will be important targets for future studies. Questions that have arisen from work shown he re are the following: 1. How does far-red affect green-light-induced shade-avoidance? 2. Is the re sponse attenuated or re latively similar in the presence of both green light and far-red waveband s? 3. How will far-red light affect flowering in the presence of green light? 4. Will there be an intermediate effect when both systems are stimulated? Another interesting extension of this work w ill be its potential influence on plant ecology, measured as the natural variation present in Ar abidopsis accessions repres enting specific regions, climates, and culture conditions. Ecotypes may vary in their green-light-induced responses. While similar trends were seen between Columbia (Col-0) and Landsberg erectus (L er ) ecotypes, differences between RB and RBG treat ments were much more apparent in Col-0 ecotypes. These differences may be due to natura lly larger rosette diameters in Col-0 ecotypes. This may allow for more variability in elonga tion and leaf hyponasty whereas more compact
70 rosette diameters in L er do not permit as much shade mani pulation. Other Arabidopsis ecotypes may show still greater variability for their respons es to supplemental green light. Previous work demonstrated that Arabidopsis accessions were quite varied for shade avoidance responses induced by low ratios of red to far-red (Botto and Smith, 2002). Ethylene (Pierik et al., 2004), gibberellins (P ierik et al., 2004) a nd auxin (Morelli and Ruberti, 2000) have all been im plicated in mechanisms underlying the shade avoidance response. Interaction between these and green-light-sen sing-pathways are also points of interest. Additionally, the transcriptome associated with induction of shade avoidance by low ratios of red to far-red has been defined (Devlin et al., 2003). It desc ribes a distinct set of transcripts affected by simulated shade (Devlin et al., 2003). Green light may affect these transcri pts, as well as those involved in anthocyanin biosynt hetic gene regulation. A better understanding of how, when, and where green-light-induced shade avoidance respons es occur will help diss ect their convergence with effects of phytochrome and blue light systems. Results of this work indicate th at the previously unrecognized effects of green light have a prominent influence on tailoring plant growth an d development to conditions set by the natural environment. However, the effects are not appare nt in plants grown under high fluence rates of white light. It is more likely that the green-lig ht-sensitive system is directing plant responses under marginal light conditions, such as foliage shade or laboratory conditions containing unnatural ratios of narrow-bandwidth LED light. This finding extends to a pr actical level, as the results suggest the reconsideration of artifici al lighting regimes for plant growth. Greenhouses and growth chambers are typically supplied with fluores cent fixtures that pr incipally emit light equivalent to the RGB conditions used in the present experiments (although fluorescent bandwidth is greater due to accessory fluors that expand the bandwidth re lative to the discrete
71 peaks seen in RGB LED treatments). Through addition or subtraction of the green light component of visible light, it is clearly possibl e to accelerate, skew, or potentially manipulate plant growth and development. In addition to greenhouse conditions, this work is relevant to densely-packed fields of crop species. Sin ce similarities were f ound between green light responses of Arabidopsis and Fragaria vesca these may extend to othe r plant species as well. Materials and Methods Plant Materials The genotypes tested are identi cal to those previously assesse d for responses to blue light (Folta and Spalding, 2001); cry1-304 cry2-1 phot1-5 phot2 (nph1-5cav1-1) and phyA201 phyB5 The phyA phyB mutant is in the L er background; all others are in the Col-0 background. Plant Growth For Arabidopsis thaliana: Plants were grown in soil in fl ats modified to fit precisely under the LED arrays (made with American Bright LEDs) with even distribution of light. Approximately 50 seeds from each genotype studied were applied to 16 cm2 of wetted soil surface using a funnel to assure loca lization of each genetic line. Each flat contained a grid of 4 x 3 genotypes. The seeds were strati fied at 4C for 72 h and then transferred to white fluorescent light for 96 h (Figures 4-1 through 43) or 1 week (Figure 4-4) to ensure that wavelength-specific effects were attributable to gr een light effects on plant development and not germination timing. Seedlings were transferred to positions under LED arrays and watered approximately every third day with saturating amounts of 0.1x commercial fer tilizer dissolved in distilled water. Plants were grown under constant irradiation to dampen the effects of the circad ian oscillator on stem elongation. The first treatment consisted of irradia tion from only red and blue LED light with a total fluence rate of ~75 mol m-2 s-1 (15 mol m-2 s-1 blue light). The next treatment consisted of same red-blue treatment supplemented with gr een light (total fluence rate ~90 mol m-2 s-1). In
72 both treatments, the average fluence rate was obta ined from sampling multiple positions in the plant-growth grid area. For Fragaria vesca : Seeds were sterilized using the fo llowing method: Seeds were covered with 0.01% Tween 20 and shaken for 15 min, rinsed with sterile diH2O, covered in 70% ethanol and shaken for 1 min, rinsed several times, then covered with 25% bleach and shaken for 10 min, then rinsed several times, place in sterile wa ter and placed on a shaker until germinated. Germinated seedlings were transplanted to soil, covered with Saran Wrap and placed under treatments, acclimated, and left under treatments fo r nine weeks. Leaf area measurements were taken by first affixing the largest leaf per plant to a piece of tape, then taking a high-resolution picture of the leaf with a size standard, and then measuring the area with ImageTool as described below for Arabidopsis. Light Sources Actinic light treatments were generated using narrow-bandwidth LED light supplied by American Bright LEDs controlled by custom electro nics as described (Fo lta et al., 2005). Light composition was assessed using an Apogee spectr oradiometer and Spectra Whiz software. The emission spectra of all light s ources are viewable on-line at www.arabidopsisthaliana.com/lightsources. Measurement Whole plants and plant organs were harveste d, and then flattened and affixed in their native positions to adhesive electrical tape. Sp ecimens were imaged with a digital camera and measurements of organ size were performed using UTHSCSA Image Tool (Version 3.0 for Windows) with comparisons to adjacent size standards. Measurement of rosette diameter was performe d using the same softwa re indicated above.
73 Anthocyanin Accumulation Tissues were either soaked in 100% Methan ol: 1% HCl directly (Fluence rate / response experiments) or first ground with liquid nitrogen (all other tissues) and then soaked overnight at 4C (wrapped in foil). The next day tubes were centrifuged at maximum speed and absorbance at 530 nm was measured. Measurements were record ed and divided by the total amount of fresh tissue used to get the absorbance at 530 nm per weight (grams), or A 530 nm / g.
74 Figure 4-1. Supplemental green lig ht induced a shade response. Wild-type (Col-0) Arabidopsis plants were grown under red and blue LED light (RB; Red light~65 mol m-2 s-1, Blue light ~15 mol m-2 s-1) or identical RB conditions s upplemented with green light (RGB; same as RB with ~15-20 mol m-2 s-1 Green light) for 15 d. Conspicuous differences in leaf area, petiole length and leaf hyponasty were observed under RGB, responses typically indicative of low-light conditions or a decreased red to far-red ratio. The ratio of red to far-red light was identical between conditions (with negligible far-red detected).
75 Figure 4-2. Supplemental green light induced petio le elongation and inhibited leaf expansion. A) Individual wild-type rosettes from a singl e representative plant grown under RB or RGB were dissected and conspi cuous leaf attributes were measured. B) Petiole length divided by total leaf length. C) The m ean leaf blade area from leaves. The measurements in B and C were derived from the second leaf from at least 30 individual plants. Error bars represent standard error of the mean. Bar = 5 mm.
76 Figure 4-3. Supplemental green light effect s are maintained in photoreceptor mutants. The effect of green light was tested in photoreceptor mutants, plants lacking functional genes encoding known photoreceptors for red, blue and far-red light. The phyA phyB and cry1 cry2 mutants were grown unde r RB and RGB, their rose ttes were dissected and leaf attributes quantified. A) Leaf whorls from a single representative phyA phyB plant grown under RB or RGB conditions. B) The average petiole length relative to total leaf length and C) Mean leaf blade areas. All measurements were obtained from the second leaf of at least 19 individual plants. Error bars represent standard error of the mean. Bar = 5 mm.
77 Figure 44. Green light res ponses are fluence-rate-dependent. Green light was added at 0.5, 4.5 and 15 mol m-2 s-1 to a constant background of RB, as in Figure 4-1 and physical attributes of plants were photographed and quantified. A) A dose-dependent change in leaf hyponasty, petiole lengt h and leaf area is observed in representative plants. B) The mean petiole length as a function of total leaf length measured from at least 15 plants, except in the 0.5 G treatments wher e only 6 plants were measured. C) The average leaf blade area (derived from the second leaf).
78 Figure 4-5. Anthocyanin accumulation under increas ing amounts of supplemental green light is visible as purple color near the base of the petiole for plants grown under RB, RBG (G=4.5 mol m-2 s-1) and RBG (G=15 mol m-2 s-1).
79 Figure 4-6. Anthocyanin accumulation decreased unde r supplemental green light for wild type (Col) plants (purple) and cry1 cry2 mutants (green) when leaves (including petioles) were examined after RB or RBG treatments. Anthocyanin levels are represented as the absorbance at 530 nm/ fresh weight (in gr ams). Data represent at least 5 leaves plus petioles per treatment a nd at least 2 experimental repl icates. Error bars represent the standard error of the mean.
80 Figure 4-7. Vegetative features of Fragaria vesca after 9 weeks growth under RB (grey columns) and RGB (purple columns) light environments. From left to right, column 1 shows the average number of leaves per plan t, column 2 shows the average number of fully differentiated leaves (3 leaflets/ petiole) per plant, column 3 shows the average number of fully-differentiated leaves per to tal number of leaves per plant, column 4 shows the average number of runners per plant, and column 5 shows the average length (in cm) of the longest petiole per plan t. Error bars represent the standard error of the mean.
81 CHAPTER 5 CANDIDATE GENE APPROACH TO IDENTIFY GREEN LIGHT PATHWAY COMPONENTS Introduction Light is a critical signal th at guides developmental decisi ons in dark-grown seedlings. Effects of red, blue, and far-red light in regula ting molecular, physiological, and biochemical events during seedling development have been we ll-described. When compared to red and blue light pathways, green light pa thways are less well-defined. Evidence of green light opposing norma l photomorphogenic progression has been presented (See Chapter 2). However, many of th ese experiments were li mited by light sources and light measuring equipment. Recent work examining seedling responses to green light substantiates historical findings. Folta (2004) demonstrated that a short pulse of dim green light induced an increase in hypocotyl elongation. Th is response followed the Bunsen-Roscoe Law of Reciprocity and was present in a background of dim-red light. In addition, green-light-induced responses were present in all photoreceptor mutants tested indi cating that this green light response may be sensed through a novel photorecepto r. Also, Zeiger and colleagues (Frechilla et al., 2000) showed that green light negates blue-i nduction of stomatal openi ng and Talbott et al. (2003) indicated that this respons e requires NPQ1. Also, Dhingra et al. (2006) demonstrated that green light induces a decrease in plastid tran script accumulation which again opposes red and blue induction. In addition to such evidence from the literature, experiment s within Chapter 3 of this work demonstrate green lig ht opposition to red and blue inhibition of hypocotyl elongation under combinatorial light treatments. Together these experiments de monstrate a negative role for green light in normal light-indu ced phenotypes in early develo pment. Although complete greenlight signal transduction pathways for these responses have not been described, some green light responses have been linked to cr ys (Bouly et al., 2007; Chapter 3 of this work), while others are
82 cry-independent (Folta, 2007). These discrepanc ies indicate there are multiple green light photoreceptors. To elucidate green light path way components, a candidate ge ne approach was implemented and experiments are described herein. Examina tion of Arabidopsis genomic sequence led to identification of several genes with high sequenc e similarity to visual cycle components. One gene had high sequence similarity to Rpe65 a gene necessary for chromophore synthesis/modification in animals ( Max4 / Ccd8 in plants) (Baylor, 1996 ; Moiseyev et al., 2005). The max m ore a xillary branching, mutants were isolated in a screen for regulators of lateral branching (Sorefan et al., 2003). Two of these mutants, max4 and max3 ( ccd8 and ccd7 respectively), cleave multiple carotenoids in E. coli and are thought to act in the same branching pathway in plants (Booker et al ., 2004; Schwartz et al., 2004). Experiments indicate that the cleavage product of CCD7 and CCD 8 is likely to be a mobile regulator of auxin transport (Schwartz et al., 2004; Bainbridge et al., 2005; Bennett et al., 2006) Expression data show that CCD8 is localized to the chloropl ast (Auldridge et al., 2006). In animals, RPE65 is required for conversion of the all-trans retinal to 11cis retinol, the preferred chromophore in animal vi sion (Baylor, 1996, Moiseyev et al. 2005). Although retinoids, specifically, all-trans retinal have been identified in plants (Lorenzi et al., 1994), a formal function has yet to be described. Roles for retinal-based receptors have been identified in other organisms such as Pelvetia (Robinson et al., 1998), and have light sensor roles in this species. CCD8 is thus an interesting candidate for contribution to photosensing or signaling mechanisms in plants. Experiments described in this chapter ex amined green-light-induced responses in ccd8 mutants, using previously established conditions that demonstrate green light responses (Folta
83 2004, Dhingra et al. 2006). Results demonstrated that CCD8, whether involved in sensing or subsequent responses, is requi red for green-light-induced dow n-regulation of chloroplast transcript levels and may be involved in long-term green-light-i nduced stem elongation. Results Isolation and Characterization of T-DNA Mutants For this study, T-DNA insertion lines were obt ained through the Salk Institute. Lines used were homozygous and verified both by PCR a nd by the phenotypic loss of apical dominance characteristic of homozygous ccd8 mutants (Auldridge et al., 2006). The T-DNA insertion locations are shown in Figure 5-1. Hypocotyl Elongation Dark-grown 2-d-old mutantand wild-type -seedlings, were transferred to narrowbandwidth, monochromatic green light treatme nts and evaluated after 3 d (72 hours). The ccd8 mutants exhibited light hypers ensitive phenotypes such as shorter hypocotyls and more open, expanded cotyledons relative to wild type seedlings (Figure 5-2). If this hypersensitive response is specific to green light, then one interpretation of this result is that CCD8 is required for normal green-light-induced hypocotyl elongation. Although green light specificity was not examined in thes e experiments (i.e. comparing mutant and wild type under other wavelengths to account for ge neral light sensitivity) mutant and wild type seedlings were the same length in the dark, indicating an effect on light-mediated stem elongation and not simply a de fect in elongation growth. Because these mutants displayed aberrant gr een light phenotypes, they were examined with finer resolution as describe d by Folta (2004). In this report a brief pulse of green light induced a transient increase in hypocotyl elongati on rate (Folta, 2004). In the present work, the ccd8 mutants responded normally to green light (Fi gure 5-2) (Folta, 2004). The data indicate that
84 CCD8 is not required for stem elongation in 48-h-o ld seedlings exposed to monochromatic green light. Chloroplast Transcript Regulation In addition to effects observed on hypocotyls in early development, green light induced decreases in plastid transcripts, which is oppos ite to red and blue induction (Dhingra et al. 2006). Chloroplast transcript accumulation was exam ined for mutants and wild type seedlings after a brief pulse of green light as defined in Dhingra et al. (2006). In wild type seedlings, a brief pulse of green light down-regulated chloroplas t transcript accumulation as in Dhingra et al. (2006). In the ccd8 mutants, an increase rather than decr ease was observed in plastid transcript abundance (specifically psaA and psbD ) following a brief pulse of green light ( psaA : Figure 5-4; data not shown for psbD ). Gene expression experiments in Figure 5-4 indicate that ccd8 mutants do not generally show aberrant light-regulated gene responses Levels of ELIP mRNA, a phytochrome-induced nuclear-encoded transcript, are up-regulated in both ccd8 mutants and wild type seedlings following a brief pulse of green light. In microa rrays described by Dhingra et al. (2006), ELIP and some other phy-induced transcripts were up-re gulated following a brief pulse of green light. This was expected since green light stimulates phytochrome and indicates that differences between mutant and wild type in gene regulatio n are specific to green light (Figure 5-4). Pharmacological Studies Since CCD8 is required for green light aff ects on chloroplast transcript regulation, the next experiments were designed to test the role of CCD8 in this response. Pharmacological experiments were conducted based on the ro le of RPE65 in vision. Affects of exogenous all trans retinal (ATR), a put ative product of CCD8 activity and potential chromophore, on green light chloroplast transcript regulation were evaluated. The ccd8 mutant seedlings were grown in
85 darkness in the presence of micromol ar and sub-micromolar amounts of all-trans retinal (ATR). Chloroplast transcript accumulation was obser ved after dark or green light treatments. Micromolar and sub-micromolar amounts of all-trans retinal were able to partially rescue greenlight-induced down-regulation of chloroplast tran scripts; however, a complete dose-response was not observed over the range of con centrations tested (Figure 5-5). Discussion Candidate genes were identified through seque nce analysis and tested for green-lightinduced responses including green -light-induced stem elongation in 2-d-old seedlings, long-term stem elongation (72 h), and down-regulation of chloroplast transcript accumulation. The ccd8 mutants were aberrant for long-term stem el ongation under monochromatic green light and for green-light-mediated down-regulation of plastid transcripts. These experiments suggest that CCD8 may play a role in long-term, green-light -induced stem elongation. However, it is also possible that light hypersensitivity in general, i.e. to all waveba nds, could be the culprit of the advanced light phenotypes observed in the mutant To answer this question, affects of other wavebands on long-term stem elon gation should be examined. The ccd8 mutant growth kinetics showed wild type trends when exposed to a br ief pulse of green light. CCD8 is not required for this early green light stem response. As in ot her light sensing and si gnaling regimes, CCD8 may be involved with some, but not all, green-light-induced responses. When chloroplast transcript accumulation was examined, the ccd8 mutants did not behave as did wild type seed lings. Rather than wild-type, gr een-light-mediated down-regulation of plastid transcripts, tran script levels increased in ccd8 mutants following a brief pulse of green light. This response suggests there may be an underlying antagonism between phytochrome stimulation of psaA and green light repressi on, and that CCD8 is requir ed for this response.
86 Application of micromolar a nd sub-micromolar amounts of all-trans retinal (ATR), a potential chromophore, partially re scued wild type green light re sponses. Partial rescue of the wild type response may be the result of the deliv ery method used in these experiments. Addition of ATR to media may not allow efficient uptake by seedlings. Also, ATR is hydrophobic, toxic to plants, and light-labil e. Therefore, when these plates were exposed to light, for the purpose of synchronizing germination, the ATR might have started breaking down a nd therefore may not have been readily available to seedlings. One im provement to this procedure would be to spray plants with all-trans retinal after 2 d growth, and before the green light pulse. Another possible reason for partial rescue and lack of a dose-response is that ATR ef fects are not specific to green light and can cause a more gene ral plant stress response. Although this is a possi bility, it is unlikely, since sub-micromolar amounts induced at least pa rtial responses. Complementation tests using w ild type dog or mouse versions of CCD8 may help resolve CCD8s role in green-light-induced down-regulat ion of chloroplast tr anscripts because the chemical activity for CCD8 has been well establis hed in these organisms. It will also be interesting to evaluate ccd8 mutants for other green-light-induced responses as indicated in Chapter 3 and 4 of this work. Also, CCD7 has b een linked to the same branching pathway as CCD8 (Sorefan et al., 2003; Booker et al., 2004; Schwartz et al., 2004). Observation of ccd7 mutants and ccd7 ccd8 double mutants under these conditions is also of intere st, since CCD7 and CCD8 are in the same lateral branching pathway. Materials and Methods Plant Lines Two homozygous T-DNA insertion mutants we re used in this study (SALK_082552 and SALK_072750). They were isolated from the Salk Institute Genomic Sequence Library and TDNA insertion points are indi cated in Figure 5-1.
87 Photomorphogenic Development Seeds were surface sterili zed and placed on minimal media containing 1mM KCl, 1mM CaCl2, and 1% DIFCO agar. The plates were verna lized for at least 48 h at 4C, and then positioned vertically under treatments. Following 48 h in a 37C incubator, plates were placed in conditions and monitored under treatments. Light treatments were generated using fluorescent light and colored filters. For Fi gure 5-2, seedlings were imaged after 72 h treatment with an 8.0 megapixel digital camera. Transcript Analysis 2-d-old dark-grown seedli ngs were exposed to a brie f pulse of green light (100 mol m-2 total fluence), wrapped in foil, and moved to da rk for one hour. At the end of 1 h, plates were dropped into liquid nitrogen. RNA extractions we re done following the Qiagen RNAeasy Plant Mini Kit. Northern blots were probed with randomly primed 32P PCR products. Retinal Treatments The all-trans retinal (from Sigma-Aldrich, St. Loui s, MO) was re-suspended in ethanol and the concentration was determined spectrophot ometrically. For long-term growth experiments all-trans retinal was added directly to cooled mini mal media and dispensed into plates under dim red light.
88 Figure 5-1. Insertion sites for the ccd8 T-DNA lines obtained via the Salk Institute and evaluated in this study. Blue boxes represent exons. Figure 5-2. The ccd8 mutants are defective for green -light-induced stem elongation. Representative seedlings of ccd8 mutants (left) and wild t ype (Col) (right) are shown after 72 h of growth under green light (20 mol m-2 s-1). SALK082552 SALK072750
89 Figure 5-3. The ccd8 mutants demonstrate green-light-i nduced hypocotyl elongation in 2-d-old seedlings. Stem elongation relative to dark rate (1) is shown on the Y-axis. The Xaxis represents time (min) following treatment (positive values) and time before treatment (negative values) or time at treat ment (time 0). Green light was given at time 0. At least 20 seedlings for each genotype were evaluated with 2 experimental replicates.
90 Figure 5-4. The ccd8 mutants were aberrant for green -light-mediated down-regulation of chloroplast transcripts ( psaA ) but wild type for phy gene regulation. A) Northern blots probed with psaA after either dark (D) or pulsed green light (G) treatments for Col and ccd8 mutants. Ethidium stained agarose gels show 18S levels. B) Quantified data from 5 or more biological replic ates. C) Northern Blot of Col and ccd8 mutants probed with ELIP (a phytochrome-induced, nuc lear-encoded transcript) after dark (D) or green light (G) treatments. A B C
91 Figure 5-5. Partial rescue of gr een-light-mediated down-regulation of chloroplast transcripts in ccd8 mutants with micromolar and sub-micromolar amounts of ATR. The psaA transcript accumulation is shown following a brief pulse of green light (G) or dark (D) treatments expresse d relative to dark levels. Increasing amounts of all-trans retinal (ATR) are indicated. The Y-axis indi cates the average of several (when error bars are present) experiments normalized to dark levels. .
92 CHAPTER 6 FUTURE GREEN LIGHT RESEARCH Experiments herein demonstrate specific ro les for green light in shaping seedling and mature plant growth and development. All gr een-light-induced effects demonstrate a negative role for green light in de-eti olation. Tunable, narrow-bandwid th LEDs (light emitting diodes) were used in these experiments and provide cond itions with which to te st green light effects going forward. In early seedling development, green light induced an increase in stem elongation in the presence of red and blue light at low fl uence rates which was dependent on cryptochromes. Preliminary data indicate that green light seems to delay true leaf development. These parameters should be addressed in future experiments. In addition to early development, green-light -induced responses were identified in mature plants. Green light added to a background of red and blue light induced symptoms associated with shade-avoidance syndrome. In these experiments, increased light (RGB) caused lower-light phenotypes. Supplemental green light induced an incr ease in petiole elonga tion, decrease in leaf area, leaf hyponasty, and decrease in anthoc yanin accumulation. Preliminary experiments indicate that green light may al so delay flowering. Shade-avoidance responses occurred in a dose-dependent manner and were present in al l photoreceptor mutant s tested. An obvious extension of this work is in isolation of green light pathway components. Experiments described here provide the conditions with which to obser ve mutants. Interesti ngly, diploid strawberry, Fragaria vesca, displayed some similar trends when compared to Arabidopsis plants grown under supplemental green light. Consistent with Arabidopsis trends, strawberry plants under RB appeared healthier in general when compared to plants under RGB in preliminary experiments. Also, supplemental green light induced a downregulation of anthocyanin accumulation as in Arabidopsis. Plants under RB had mo re runners per plant and more fully-differentiated leaves
93 than RGB treated plants. Strawbe rry experiments should be repeat ed and more species should be examined for green-light-induced responses going forward. By understanding how plant sense and respond to green wavebands, improvements in crop production and greenhouse growth can be made. Because these wavebands for the most part induce lower-light phenotypes, manipulation of light environments (via s upplemental or reduced green light depending on species of interest) will allow for increased desired effects.
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102 BIOGRAPHICAL SKETCH Stefanie Maruhnich was born in Durham, No rth Carolina and shortly after moved to Aliquippa, Pennsylvania, and late r to Destin, Florida where she spent most of her formative years. Upon graduating from Fort Walton Beach High School in Fort Walton Beach, Florida, she received several scholarships to the University of Florida where she began undergraduate studies majoring in botany. She was accepted into Maria Gallos lab as an undergraduate research assistant. The summer before receiving her B.S. in botany, she was awarded a research internship in Harry Klees laboratory at the University of Florida. The following August she began the Plant Molecular and Cellular Biology Program in Kevin Foltas laboratory.