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! ;; Acknowledgements I would like to thank my committee members, Dr. Clore, Dr. McCord, and Dr. Beulig for taking the time to read my t hesis and sit on my committee. Special thanks to my advisor Dr. Clore for giving me a glimpse into the world of primary science research and guiding me through the thesis process, all the while being extremely supportive and encouraging. You could not have been a better teacher or mentor to me. I would also like to thank T. Asami (University of Tokyo, Japan) for the generous gift of Brz2001. Thank you M. Tatum who conducted several replications of the wheat leaf unrolling assay and worked out some of the p rocedures as adapted from Wada et al. (1984), and who also prepared and analyzed some of the Brz2001 samples prior to her departure. Thank you Mr. Joel Thurmond for all of your assistance in the lab. Thank you to my parents, Linda and Larry Goodman, for t heir endless love and encouragement. Thank you to my family, friends, and roommates for having confidence in my efforts and for always listening to me talk about it all, especially my thesis (even when you didn't understand). I could not have done it witho ut your support. Thank you New College.
! ;;; Table of Contents Acknowledgements . . . . . . . ii Table of Contents . . . . . . . iii List of Figures and Tables . . . . . . v Abstract . . . . . . . . vi 1. Introduction . . . . . . . 1 1.1 Types of organ fusion in plants . . . . 2 1.2 C. roseus gynocial development . . . . 4 1.2.1 Significance of C. roseus carpel fusion in detai l 4 1.2.2 Stages of carpel fusion in C. roseus . . 5 1.3 Initial evidence for brassinosteroid signaling . . 10 1.4 Mutations affecting organ fusion in other species . 15 1.5 Thesis overview . . . . . . 17 2. Methods . . . . . . . . 19 2.1 Plant materials . . . . . . 19 2.2 General periwinkle bud dissection . . . 19 2.3 Fixing, dehydr ation, and embedding . . . 19 2.4 Sectioning . . . . . . . 20 2.5 Treatment with epi BL and Brz2001 . . . 21 2.6 Wheat leaf unrolling bioassay . . . . 22 2.7 Benzylaminopurine (BAP) treatment . . . 23 2.8 DNA isolatio n for use in PCR . . . . 24 2.9 Polymerase Chain Reaction (PCR) . . . 25 2.10 Cuticle permeability assays . . . . 25 2.11 Chlorophyll leaching assay . . . . 2 6 2.12 Dissecting and compound microscope imaging and digital imaging processing . . . . 27 2.13 Statistics . . . . . . . 27 3. Results . . . . . . . . 28 3.1 Treatment with Brz2001 interferes with normal carpel development . . . . . . 28 3.2 Treatment with both epi BL and Brz2001 resul ts in normal gynoecial development . . . . . 31 3.3 Wheat leaf unrolling bioassay suggests brassinosteroid like activity of carpel exudates . . . . 33 3.4 BAP does not elicit cell fate changes in abaxial cells of the carpels . . . . . . . 34 3.5 A PCR product of the predicted size amplified from C. roseus DNA using degenerate primers for BRI1 . . . 35 3.6 Cuticle permeability assays demonstrate distinct patterns of staining that change throughout gynoecial development . 37
! ;B 3.7 Results of attempts to adapt a chlorophyll leaching assay for assessing changes in cuticle permeability during ca rpel development . . . . 4 0 4. Discussion . . . . . . . . 43 4.1 Future directions . . . . . . 48 5. References . . . . . . . . 52
! B List of Figures and Tables Figure Title Page ___________________________________________________________ 1.1 Photograph of a C. roseus flower . . . . . 1 1.2 Diagram of a longitudinal section through the center of a simple flower 2 1.3 Diagram comparing fusion events in developing compound organs 3 1.4 SEM image of the carpels in the center of a C. roseus bud . . 4 1.5 C. roseus normal gynoecial devel opment . . . . 7 1.6 Background studies demonstrating a water soluble morphogen elicits cellular dedifferentiation on adaxial and abaxial carpel surfaces . 9 1.7 Structure of a b rassinolide molecule . . . . 11 1.8 Brassinosteroid signaling pathway . . . . . 12 1.9 Structure s of brassinosteroid inhibitors, spironolactone and Brz2001 14 1.10 Morphological changes in gynoecia that developed in the presence of spironolactone . . . . . 15 2.1 Diagram illustrating experimental procedure for the chlorophyll leaching assay . . . . . 26 3.1 Evidence for fusion defects in the presence of Brz2001 . . 29 3.2 Bud treated with 3 M Brz2001 . . . . . 30 3.3 Percent unrollin g of etiolated wheat leaves by epi BL, carpel exudates, and their controls . . . . . . . 34 3.4 BAP treated carpel compared with epi BL treated prefusion and fusion stage carpels . . . . . . 35 3.5 PCR product amplified using degenerate primers for BRI 1 . 36 3.6 Pattern of TBO stained carpels throughout devel opment . . 38 3.7 Pattern of ruthenium red stained carpels throughout development 39 3.8 Raw absorbance values for diffused chlorophyll over time using postfusion stage carpels . . . . . 41 3.9 Raw absorbance values for diffused chlorophyll over time using fusing stage carpels . . . . . . 42 Table Title Page ___________________________________________________________ 3.1 Assessment of fusion following treatment with epi BL and Brz2001 32
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! Chapter 1: Introduction During Catharanthus roseus ( L. ) G. Don (Madagascar periwinkle ; Fig. 1.1 ) female reproductive development, two individual carpels 1 undergo fusion approximately 13 14 days after bud initiation (Verbeke and Walker, 1985). Moreover, a d ramatic cell fate change occurs to facilitate fusion, which is an idiosyncratic characteristic of C. roseus (Verbeke, 1992b). The ontogeny of this system is well characterized; however, the mechanism s involved are not fully understood and raise interesting biological questions about organ fusion and control in cell fate. In this thesis research, a potential morphogen involved in the process and changes in carpel cuticle permeability were characterized. Prior to a detailed description of carpel fusion specif ically, various types of organ fusion in plants will first be described. Fig. 1.1 Close up photograph of a C. roseus flower (Wikimedia Commons) !!!!!!!!!!!!!!!!!!!!!!!!!!!!!! !!!!!!!!!!!!!!!!!!!!!!!!! !#$%&'()!$%'!*+'!',, &%./0123,! )*%01*0%')!23!$!4(.5'%!$)!)''3!23!62,7!"787
! 8 Fig. 1.2 Diagram of a lo n gitudinal section through the center of a generic simple flower. The carpel, which is the eg g producing structure in the center of a flower, consists of three parts: the stigma, style, and ovary. Pollen produced from anthers sticks to the stigma and germinates to form a pollen tube that grows down the long style. Two sperm nuclei are de posited in an ovule within the ovary at the base of the carpel. O ne sperm nucleus fuses with the egg cell to form a zygote and the other fuses with two polar nuclei to develop triploid endosperm. Flowers can be monocarpous (containing one carpel), apocarpo us (countaining multiple free carpels), or syncarpous (containing multiple fused carpels). C. roseus is syncarpous. Gynoecium is the collective term for all of the carpels in a flower. ( https://wikispaces.psu.edu/display/Biol110HSch/Plants+IV+ +Seed+Plants +%28Flowering%29 ) 1.1 Types of organ fusion in plants Both congenital fusion and postgenital fusion occur in the development of compound organs (as reviewed in Verbeke, 1992a; Walker, 1975a). Congenital fusion occurs when a compound structure develops du e to the activity of a basal meristematic zone but the fused region is partially made up of phylogene tically different parts (Fig 1.3 a; reviewed in Walker, 1975a; Verbeke, 1992 a). Postgenital fusion (Fig. 1.3 b, c) is the union of two separately formed and distinct primordia to form a single structure and involves cell cell communication (as reviewed in Verbeke, 1992b). In the case of C. roseus two carpels in
! 9 the center of th e bud arise separately (Fig. 1.4 ) and then fuse postgenitally but the fusion zone i s spatially restricted (Fig. 1.3 c), making its distinction from congenital fusion clear. The result is a single gynoecium in the center of the bud. The union of carpels (syncarpy) is fairly common in angiosperms and is thought to improve reproductive suc cess (Walker, 1978; Verbeke 1992a). Other examples of postgenital carpel fusion occur in plants b elonging to the Ranunculaceae, Alismacrea e, Rosaceae, and Brassicaceae famil ies (Verbeke, 1992a). Syncarpy provides ovule protection from the environment, and allows ovule fertilization by pollen on different carpels and increases seed dispersal (Lolle and Pruitt, 1999). Fig. 1.3 Diagram comparing fusion events in developing c ompound organs. a) congenital fusion b) postgenital fusion c ) spatially r estricted postgenital fusion. Shaded areas represent regions that were initiated as distinct entities. Arrow (a) indicates the adaxial (inner) surface, and arrow (b) the abaxial (outer) surface of the carpel. ( adapted from Walker, 1975a)
! : Fi g. 1.4 SEM image of the carpels in the center of a C. roseus bud. The bud was dissected such that one or more sepals (S), petals (P), and anthers (A) were removed to expose the carpels (C). Bar = 100 m. Image courtesy of Dr. Judy Verbeke, used with permission. 1.2 C. roseus g ynoecial development in detail 1.2.1 Significance of C. roseus carpel fusion Typically, postgenital fusion is of a rather superficial nature (Verbeke, 1992a). C. roseus carpel fusion is unique because there are approximately 400 epidermal cells on the cont acting (adaxial) surfaces of the carpels that dedifferentiate into less specialized p arenchyma cells giving rise to solid tissue fusion (Verbe ke and Walker, 1985). This is sig nificant because usually, once a cell becomes specialized, it rarely reverts to a less specialized cell during the course of normal development (dedifferentiation) as occurs during C. roseus carpel fusion (Bruck and Walker, 1985). Furthermore, elucidating the mechanism of dedifferentiation during C. roseus carpel fusion may give insig ht into the
! ; formation of cancer cells. Although it is often thought that stem cell maturation arrest is the cause of malignant cancer cells ( Sell et al., 1993; Sriuranpong et al., 2001), dedifferentiation events have been shown to be associated with the pr oliferation of certain cancer c ells for example in the case of malignant hepatocytes and astrocytes (Sell, 1993; Moon et al., 2011; Sun et al., 2011). 1.2.2 Stages of carpel fusion in C. roseus C. roseus c arpel development is separated into three stages: prefusion, fusing, and postfusion. In the first stage, two distinct carpel primordia, which arise from the L1 L3 layers in the apical meristem, are visible with a small space separating them (Fig s 1.4 and 1.5 a; Boke, 1949; Walker, 1975a). During the pref usion stage of development (Fig. 1.5 f ) the epidermal cells divide exclusively in the anticlinal plane, are rectangular in shape and are surrounded by a thin cuticle ( Walker 1975b; Verbeke, 1992). The carpels grow to approximately 200 250 M tall before ce ll contact (Walker, 1975a; Clore et al., in revision). Upon contact, the carpels enter the fusing stage, wherein contacting epidermal cells on the adaxial surfaces (see Fig. 1.5 c) undergo adhesion and exhibit changes in t he planes o f division (Fig. 1.5 b g). Within 4.3 h of contact some cells have undergone complete dedifferentiation into isodiametric parenchyma cells and by 8.9 h fusion is complete (Verbeke and Walker, 1985). In the fusing stage, carpels are approximately 350 M tall and a distinct sutu re line is visible (Walker, 1975a; Clore et al., in revision). The postfusion stage occurs when the two carpels have completely fused together into a single gynoecium and are over about 550 M with a rounded stigma and nearly
! < undetectable suture line (Fig 1.5 c, d, h; Walker, 1975a; Clore et al., in revision). As t he gynoecium matures, the style elongates, a "skirt" develops at the base of the radially symmetrical stigma (Fig. 1.5 e), but the base of the ovary region remains unfused (Boke, 1949)
! = Fig. 1.5 Catharanthus roseus normal gynoecial development. a e Buds in different stages of development were dissected such that sepals, petals, and anthers were removed an d the remaining carpels and receptacle were photographed under a dissecting microscope. a) Prefusion carpels have not yet made contact. b) Fusing carpels have grown in contact. Arrows indicate zone of fusion. c) Early postfusion gynoecium. Again arrows ind icate zone of fusion. d) Late postfusion gynoecium consisting of stigma (SG), style (ST), and ovaries before style elongation. The distal portio n of the ovaries (DO) develops from the fusing region as well. e) A maturing gynoecium with an elongated style a nd a developing skirt (arrowhead) at the base of a now radially symmetrical stigma. f) Cross section of prefusion carpels in which the epidermal cells divide almost exclusively anticlinally (arrows indicate examples of anticlinal walls). g) Cross section o f the fusing zone in a late fusing stage gynoecium. Some epidermal cells i n the fusion plane (indicated by arrows) have new ly divided in multiple planes. h) Cross section of stigma from a postfusion gynoecium. The fusion plane (indicated by arrows) is no l onger readily visible and the cells have become isodiametric and vacuolated. Bars in a e = 200 mM. Bars in f h = 100 mM. Micrographs f h courtesy of Dr. Judith Verbeke (used with permission)
! > Pr evious studies reve aled that a diffusible unknown factor' is exchanged between carpels to elici t change s in cell fate. B arriers were placed between prefusion stage carpels which w ere permitted to develop to the postfusion stage and were then observed fo r morphological effects (Fig.1.6 a; Verbeke and Walker, 1986; Siegel and Verbeke, 1989). When the barrier was permeable, the epidermal cells on both sides of the barrier proceeded to dedifferentiate norma lly (Fig 1.6 a ), whereas if the barrier was non permeab le (e.g. if non porous polycarbonate barrier was placed between carpels), dedifferentiation did not occur (Verbeke and Walker, 1986). It was determined that the morphogen has a molecular weight smaller than 1000 by using barriers with different pore size s (Verbeke and Walker, 1986). Siegel and Verbeke (1989) also showed that t he dedifferentiating promoting factor' could be trapped in the porous barrier, which could then be moved to the abaxial (non contacting) surfaces of the carpels and induce dedifferen tiation of those (normally unchan ging) epidermal cells (Fig. 1.6 b ,c ; also confirmed in Durbak, 2004 New College of Florida thesis). The c ontacting epidermal anther cells however, were not affected by the signaling factor (Fig. 1.6 c; Siegal and Verbeke, 1989). When one of the two carpel s was rotated and grafted 180¡, the abaxial epidermal cells contacting the normal carpel also dedifferentiated (Siegel and Verbeke, 1989). Two conclusions were made from these results: 1) a morphogenetic signal is transmitt ed between the carpels to facilitate dedifferentiation and thus fusion and 2) although only the epidermal cells on the adaxial surfaces dedifferentiate during normal development, all of the carpel ep idermal cells are competent to d edifferentiate when expos ed to the unknown factor (Verbeke, 1992; Siegel and Verbeke, 1989).
! ? Fig 1. 6 a) A permeable barrier placed between carpels allows for cellular dedifferentiation during fusion to proceed, identified by increased vacuolation and ob lique plane divisions ( arrows). b) The factor' containing barrier promotes normally unchanging abaxial epidermal cells to dedifferentiate, again marked by increased vacuolation and oblique divisions (arrows). The contacting anthers did not exhi bit any changes. c ) Diagram illust rating the experimental procedure for factor trapping experiments using agar impregnated porous barriers, in which the factor was collected from between carpels and placed on the abaxial surfac es to observe cell morphology as in (b). Adapted from Siega l and Verbeke, 1989. As mentioned earlier, a thin waxy cuticle layer surrounds the epidermis, and conceivably the factor' must pass through this cuticle. The cuticle, which is a dynamic
! "@ network of cutin and waxes, protects against the external environme nt and regulates permeability of water and other small molecules by altering its composition (Lolle and Priutt, 1999; Kerstiens, 2006) It has been suggested that changes in cuticle permeability help facilitate carpel fus ion (Walker, 1975b; Lolle and Pruit t, 1999). Remnants of cuticle are visible during fusion and new cell wall material also becomes apparent (Walker, 1975c). Increased cytoplasmic activity during fusion indicates involvement of the golgi apparatus and rough endoplasmic reticulum in the deposi tion of new cell wall material and/or its enzymatic degradation (Walker, 1975c and Verbeke, 1989). T he epidermal cells lack carpel connecting plasmodesmata (cytoplasmic pathways that often interconnect plant cells) j ust af ter contact but prior to dediffere ntiation suggesting that the pathway of the morphogen proceeds throu gh the epidermal cell walls rather than through the symplast (Walker, 1975c; Lolle e t al., 1997). Van der Schoot et al. (1994) demonstrated that upon microinjection the lipid impermeable molecule, Lucifer Yellow CH, mo ves through the carpels via plasmodesmata but only after dedifferentiation had already begun. They found evidence that secondary plasmodesmata were form ed after the on set of dedifferentiation consistent with the notion that t he morphogen must initially travel through the apoplast and therefore, cross any existing cuticle (van der Schoot et al., 1994). 1.3 Initial evidence for b rassinosteroid signaling The morphogen has been largely uncharacterized. Experiments testing the classic" plant hormones (gibberellins, indole acetic acids, and cytokinins) indicated that they did not ex hibit factor like activity when applied to the abaxial surfaces of the carpels (personal communication from J.A. Verbeke to A.M. Clore; Clore et al., in revision).
! "" Likewise, the factor' was found not to be proteinaceous as evidenced by experiments in which the factor was exposed to the protease prot e inase K prior to application but retained activity (Kahn, 2006 New College of Florida thesis; Clore et al., in revision). T here is some evidence that the morphogen may be a brassinosteroid (Clore et al., in revision). This makes sense structurally, since su ch a non polar steroid (Fig. 1.7 ) should conceivably be able to move through the lipid rich extracell ular matrix. A brassinosteroid known as brassinoli de is illustrated in fi gure 1.7 Numerous brassinosteroids have bee n found to occur naturally, and have varying structures in A/B ring and side chain substituents (Yoko ta, 1997; Clouse and Sasse 1998), but brassinolide is the most abundant in plant tissue (Yokota, 1997; Clouse and Sasse, 1998). Fig. 1.7 A brassinolide molecule is depicted above with carbon numbering ( from Fujioka and Yokota, 2003). Brassinolide has a molecular weight of 480.68. Brassinos teroids, discovered in the 1970's in Brassica napus (L.) pollen (Yokota, 1997), were initially overlooked in early research of C. roseus carpel fusion because they were a relati vely new class of plant hormone (Yokota, 1997). They are involved in regulating many aspects of g rowth, including processes such as cell expansion, cell
! "8 division, vascular differentiation, and multiple aspects of reproductive and leaf development (Clouse & Sasse, 1999; Fujioka and Yokota, 2003; Karlova & Vries, 2006). Although the br assinosteroid signaling pathway is not entirely known, research has illuminated many of the associated factors involved (Fig. 1.8 ) Fig. 1.8 A diagram of the brassinosteroid signaling pathway (He et al., 2002). Unlike many steroid hormones, brassinostero ids are perceived at the cell surface and transmit signals to the nucleus via signaling cascades. Acronyms are gene products involved in the brassinosteroid signaling pathway as described in the text. In this signaling pathway ( depicted in Fig. 1.8 ) brass inosteroid perception is mediated by a leucine rich repeat transmembrane receptor kinase, BRI1
! "9 (BRASSINOSTEROID INSENSITIVE 1) (Li & Chory, 1997; Friedrichson et al., 2000 ; Ye et al., 2011 ). Coreceptors BAK1 (BRI1 ASSOCIATED RECEPTOR KINASE 1) and SERK1 (S OMATIC EMBRYOGENESIS RECEPTER LIKE KINASE 1) assist BRI1 in conveying the brassinosteroid signa l (factors not shown in Fig. 1.8 ; Karlova & Vries, 2006). Brassinosteroid binds to the extracellular domain of BRI1, causing conformational changes that prompt p hosphorylation and therefore activation of the receptor (Karlov & Vries, 2006). BRI1 and its coreceptors form a multimeric complex, which negatively regulates BIN2 (BRASSINOSTEROID INSENSITIVE 2; He et al., 2000; Karlova & Vries, 2006). Subsequently, the n uclear proteins BES1 ( BRI1 EMS SUPPRESSOR 1) and BZR1 (BRASSINAZOLE RESISTANT 1) become dephosphorylated and bind DNA (along with some additional proteins) to propagate the brassinosteroid growth responses ( Ye et al., 2011; He et al., 2000; Karlova & Vrie s, 2006). As a feedback mechanism, BES1 and BZR1 additionally inhibit brassinosteroid biosynthesis (Ye al., 2011; He et al., 2000). In the absence of brassinosteroid, the inactive plasma membrane receptors are recycled by endosomes (Karlova & Vries, 2006). The kinase BIN2, no longer inhibited by BRI1, phosphorylates BES1 and BZR1, which are then degraded by the proteasome (He et al., 2000; Karlova & Vries, 2006). Additional known factors not d iscussed in this text have also been implicated in this signaling pathway (Fig. 1.8 ; Ye et al., 2011) The hypothesis that brassinosteroid signaling may play a role in C. roseus carpel fusion has been also supported by some recent research (Kahn, 2006; Clore et al., in revision). Exogenous brassinosteroid was found to e licit cell fate change on abaxial surfaces of carpels similar to that which occurs with carpel exudates application (Kahn,
! ": 2006; Clore et al., in revision). Furthermore, treatment with the b rassinosteroid inhibitors, spiro nolactone and the newer and more s pecific inhibitor Brz2001 ( Fig. 1.9 ; Sekimata et al., 2001; Asami et al., 2004) interfe res with normal carpel fusion (Fig. 1.10 ; Kahn, 2006; Clore et al., in revision). Mature gynoecia treated with brassinosteroid inhibitor displayed uncharacteristic morp hologies including a split skirt and a bilaterally lobed stigma, and although superficially adhered, were easily separable upon minimal manipulation wi th a dissecting needle (Fig. 1.10 a, c; Kahn, 2006; Clore et al., in revision). Therefore, it appears that superficial fusion proceeds despite treatment with spironolactone or Brz2001 Fig 1.9 Structures of brassinosteoroid inhibitors sprironolactone (left ; Wikimedia Commons ) and the newer more spe cific inhibitor, Brz2001 (right; Sekimata et al, 2001).
! "; Fig. 1.10 Morphological changes in gynoecia that de veloped in the presence of spiro nolactone demonstrated perturbed gynoecium development. a) A nearly mature gynoecium treated with 150 M spirinolactone displays a split skirt (arrow) and a bilaterall y lobed stigma. Similar effects were found when treated with 5 10 M B rz2001 b) In comparison, the EtOH treated control shows a normal gynoecium with an intact skirt and radiall y symmetrical stigma. c) A spiro nolactone treated gynoecium probed with a diss ecting needle split easily into two equal halves. Untreated or EtOH treated control gynoecia at that age do not separate when manipulated with a dissecting needle, and instead normally results in mangled stigma detached from their stigmas (not shown). Bars = 500 M 1.4 Mutations affecting organ fusion in other species Studies utilizing Arabidopsis thaliana (L.) Heynh. mutants provide insight into the relationship between the cuticle and organ fusion (Lolle et al., 1997; Sinha, 1998; Weng et al., 2010). A m utation in the FIDDLEHEAD gene causes epidermal cells to undergo contact mediated fusion responses between various tissues (such as any leaves that come in contact with one another), as well as ectopic carpel pollen interactions (Lolle et al., 1997). The c ontact mediated fusion is similar to that of C. roseus carpel fusion in that it is postgenital but it is more superficial and does not exhibit change s in cell fate ( Lolle et al., 1992; Lolle et al., 1997). The FIDDLEHEAD gene product is involved in the sy nthesis of long chain fatty acids relating it to the FATTY ACID ELONGATION family of proteins, likely in the class that includes ketoacyl CoA synthase (Pruitt et el., 2000 and Yephremov, 1999). This finding raised the possibility that the fiddlehead ( fd h 1 ) mutant's change in lipid composition may affect the permeability of its cuticle, prompting organ adhesion between ep idermal cells (Pruitt et al., 2000). Cell wall
! "< fractions of fdh 1 indeed revealed differences in lipid composition consisting of increa sed long chain, high molecular weight fatty acids and decreased low molecular weight fatty acid chains (Lolle et al., 1997). U sing in situ hybridization, expression of t he FIDDLEHEAD gene was shown to occur in epidermal tissue (Lolle et al., 1997; Pruitt e t al., 2000 ). In addition, the chlorophyll can be leached from mutant tissue more readily than in the wild type, which further supports the hypothesis that cuticle modification alters its permeability to facilitate fusion (Lolle et al., 1997). Additional examples of mutations in Arabidopsis and Zea mays (L.) demonstrate that cuticle modification is important in organ fusion. The Arabidopsis cer1 cer2 cer3 cer6 and cer10 mutants are deficient in cuticular waxes (Lolle et al., 1997; Pruitt et al., 2000 ). The CER (ECERIFERUM) proteins are involved in the conversion of long chain aldehydes to alkanes in various steps of cuticular wax bio synthesis and is important in contact mediated interactions between carpel and pollen, which fail in cer mutants ( Aarts et al., 1995; Pruitt et al., 2000). Mutations in the LACS (lo ng chain acyl CoA synthetases ) genes prevent l ong chain acyl CoA formation again contributing to alterations of cuticular waxes and cutin in the epidermal cells of Arabidopsis (Weng et al., 2010). In M aize, adherant1 ( ad1 ) mutants show inappropriate organ fusion throughout development, as early as in the ungerminated embryo (Sinha, 1998; Pruitt et al., 2000 ). Although the mutation does not affect cell fate, cuticle composition and cell wall are modi fied (as reviewed in Sinha, 1998). These examples collectively demonstrate how changes in cuticle composition influence cuticle permeability and promote organ fusion. Based on these other reports, it has been hypothesized that during C. roseus gynoecial de velopment, changes in cuticle permeability may promote fusion, perhaps by increasing
! "= adhesivity and/or by allowing passage of the factor' (Verbeke, 1992a ; Lolle and Pruitt, 1999 ). 1.5 Thesis overview For this thesis research, evidence for brassinosteroid signaling and changes in cuticle permeability during C. roseus carpel fusion were investigated. Preliminary evidence for brassinosteroid sig naling during carpel fusion had already potentially contributed to the character ization of the once mysterious fact or' that prompts dedifferentiation (Kahn, 2006; M. Tatum, unpublished preliminary data). Research was conducted for this thesis to continue to test the hypothesis t hat brassinosteroid signaling is involved First s ections were made of carpels from buds th at had been treated with the brassi nosteroid inhibitor Brz2001 to see if such treatment perturbed carpel development histologically and to see whether the results were consistent with those using the older and less specific inhibitor, spironolactone ( Kahn, 2006; Clore et al., in revision) A control experiment involving treating cuttings with both Brz2001 and 24 epibrassinolide (epi BL) was conducted to see if the addition of brassinosteroid could r escue' carpel morphology. Such a control would help to con firm specificity of the inhibitor, proported to inhibit brassinosteroid synthesis (Sekimata et al., 2001). W heat leaf unrolling bio assays (Wada et al., 1984) were conducted so that carpel exudates could be tested for bra ssinosteroid like activity. In addit ion, C. roseus DNA was isolated and degenerate primers were used in attempt to amplify the BR1 gene, which encodes the brassinosteroid receptor (Li and Chory, 1997; Ye et al., 2011) If successful, this could both help to validate the notion that brassinos teroid signaling is involved and could also serve as an important starting point for future studies. Finally, the abaxial epidermal cells fusing
! "> stage buds were treated with a cytokinin, called benzylaminopurine (BAP) This hormone reportedly does not mimi c the factor' (J.A. Verbeke personal communication to A.M. Clore), but our manuscript reviewers (Clore et al., in revision) wanted us to test it directly since it is known to promote cell division in many tissues (Werner et al., 2001). To test the hypoth esis that the cuticle becomes modified during fusion, toluidine blue and ruthenium red permeability assays were employed to assess changes in cuticle permeability through out different stages of gynoecial development. These assays are based on the premise t hat these two dyes bind cell wall components (Curvers et al., 2010), and only stain appreciably if the cuticle is modified to become more permeable ( Tanaka et al., 2004; Viosin et al., 2009 ). Finally, a chlorophyll leaching assay (Lolle et al., 1997) was e xplored for potential use in comparing the chlorophyll diffusion rates in different stages of carpels
! "? Chapter 2: Methods All chemicals were purchased from Sigma Aldrich (St Louis, MO, USA) unless otherwise stated. 2.1 Plant materials Periwi nkle ( Catharanthus roseus ( L.) G. Don ) plants were maintained at the New College of Florida greenhouse in Sarasota, FL with supplemental lighting to give a 16h/8h light/dark photoperiod The plants were watered 2 3 times daily and fe rtilized twice weekly using Southern Ag Powerpak 20 20 20 solution. 2.2 General periwinkle bud dissection All dissections were performed under a dissecting microscope. Buds were harvested from the greenhouse and dissected in a petri dish containing filter paper moistened wi th deionized (dI) water. Size 11 scalpels were used in one of two ways to access the carpels: sepals, petals and anthers were removed exposing the carpels at the center of the bud, or a window was excised in the petals and one or two anthers removed, revea l ing, the carpels within surrounding tissue. This latter method was used for in vivo treatment s (i.e., treatments of the carpels while still on the plant). 2.3 Fixing, dehydration, and embedding T o observe cellular morphology changes using light microsco py after hormone application, plant tissue was prepared by the following process. Buds were first fixed in 4% formaldehyde, 1% gluteraldehyde (4F1G) and incubated for a minimum of 4 h on a rotator. After the fixative was decanted, buds were rinsed in phosp hate buffer saline
! 8@ (PBS ; 0.1 M phosphate buffer with 0.0025 M potassium chloride and 0.137 M sodium chloride at pH 7.4 ) three times for 10 min and subsequently dehydrated using an ethanol series of 20, 30, 40, 50, 60, 70, 80, 90, (v/v) and (3x) 100 % for 1 5 min each. Next, LR White resin (Ted Pella, Inc. Redding, CA, USA ) was added to the ethanol containing the tissue in increments using the following LR White to ethanol ratios: 1/3:2/3, 1/2:1/2, and 2/3:1/3 and were allowed to rotate for a minimum of 4 h each. The liquid was then removed and pure LR White was added to the tissue, which was incubated for # 4 h (up to overnight) on a rotator, followed by two additional pure LR White changes 4 h each. To embed the tissue, LR White was mixed with the catalys t benzoyl peroxide in a 500g /9.9g ratio and placed on a rotator for approximately 1 h to mix thoroughly. B uds were then placed in BEEM capsule, with catalyzed LR White and inc ubated in a vacuum oven at 60 ¡ C for 24 h to polymerize. I prepared BAP treated samples using this method, which was also used by M. Tatum for the preparation of Brz2001 treatment series. Brz2001 sample buds were treated with a range of concentrations from 1 15 M of the inhibitor (Brz2001) or concentrations of DMSO corresponding to t he amounts used to deliver the inhibitor for controls. They were sectioned (see section 2.4) and analyzed by myself and M. Tatum as detailed in the Results section. 2.4 Sectioning T o anal yze the Brz 2001 samples made by M. Tatum (2008) and BAP treated bu ds tha t I prepared (see section 2.7 ), LR white embedded tissue was trimmed Blocks were removed from their outer gelatin capsules and mounted onto metal chucks. Block faces
! 8" were carved into a pyramidal shape with a flat trapezoidal face u sing a single edg ed razor blade for rough cuts or a double edged blade for subsequen t precise shaping B locks in chucks were then placed into the Sorvall MT2B Ultramicrotome for sectioning. Sections were cut to 1 2 m thick using glass knives in automatic and manual advan ce modes Sections were transferred onto water (dI) filled boat s and smoothed using a heat pen. Next a Perfect Loop¨ (Electron Microscopy Sciences Inc., Hatfield, PA, USA ) was used to pick up indiv idual sections to be placed on glass slide s The sections w ere dried on a hot plate on low heat for a few seconds, stained briefly with 0.33% (w/v) methylene blue i n water, and then rinsed with dI water. They were then covered with Permount¨ and coverslipped. 2.5 Treatment with epi BL and Brz2001 To assess the a bility of exogenous brassinolide to rescue the effects of the brassinosteroid inhibitor Brz2001 stem cuttings, containing clusters of prefusion and fusion stage buds were treated with 10 M Brz2001 (courtesy of T. Asami, University of Tokyo, Japan) and 1 M 24 epibrassinol ide Similar to the method previously used by M. Tatum (2008) for treatment with Brz2001 alone, 22 ml of 10 M Brz2001 and 1 M epi BL in tap or dI H 2 O were added to scinti llation vials covered with foil A ir was bubbled into the vials e very few days using a pasteur pipet for aeration. C uttings were maintained in a growth chamber at 25 ¡ C, with a16h/8h light/dark photoperiod (except for one attempt in the greenhouse see results for explanation). A fter you ng buds developed to early post fusi on or a more mature gynoecium, they were dissected to reveal the carpels (as described in section 2.2 ) which were manipulated with a dissecting needle (by
! 88 lowering slowly through the gynoecium in the apical to basal direction) to test the integrity of the fusion plane and ; therefore to determine whether exogenous brassinosteroid rescued the effects of the inhibitor Brz2001. 2.6 Wheat leaf unrolling assay The wheat leaf unrolling bio assay was used to test carpel exudat es for brassinosteroid activity, adapt ed from Wada et al. (1 984) with several adjustments. The extent of w heat leaf unrolling stimulated by exudates secreted by carpels was compared with that of epi BL (positive control) Apogee dwarf wheat (courtesy of B. Bugbee, Utah State University, USA) w as germinated in moist paper towels for 24 hours in darkened chamber before being planted into pots with wet vermiculite. They were allowed to grow for an additional 6 days in the dark prior to use in the assay. Carpel exudates were obtained from fusing ca rpels incubated for 72 hours (at 25 ¡ C in a Powers Scientific $ growth chamber with a 16/18h light/dark cycle) on plates containing 0.8% (w/v) low melting point agarose (Sigma Aldrich, St Louis, MO, USA ) dissolved in sterile distilled water or absolute ethanol. For each sample, five fusing carpels were excised and place d horizontally in a 5 mm area of the plate After the carpels were carefully removed, agar plugs that were in contact with the carpels were excised and added to a 1.5 ml microfuge tube con taining 750 l MAB ( 2.5 mM maleic acid buffer pH 7 ; Wada et al., 1984). Positive control samples were prepared as follows. E pi BL dissolved in ethanol was added to 0.8% (w/v) agar to a final concentration of 0.01 M/ml. E thanol or water was used for the ne gative control plates A gar plugs were removed for the various control plates and added to a microfuge tube with 750 l MAB.
! 89 T ubes were placed on a rotator platform for 1 hour at medium speed and subsequently centrifuged on high speed (~14,000 x g) for 15 seconds. Droplets of supernatant were placed in small weigh boats lined with parafilm in the dark under a dim green safelight Segments of etiolated wheat leaves were then balanced on the droplets and incubated for 24 hours at 30 ¡C in total darkness. The diameter of each segment was measured u sing a digital caliper (Fisher S cientific $ ) before and after incubation, along with the width of the fully unrolled leaf segment. Percent of wheat leaf unrolling was then calculated using the following formula: Eq. 2. 1 W s W i W F W i 100 where W i is the initial diameter of the segment, W s is the diameter of the leaf segment after 24 h of incubation, and W F is the diameter of the fully unrolled leaf segment. A Mann Whitney U test was performed to compare the co ntrols performed by M. Tatum and myself in order to account for any variables in the tissues or conditions since the time of M. Tatum's research although the same procedures and variety of wheat were used. The results were combined w ith those obtained by M. Tatum and analyzed using a Mann Whitney U Tes t (see section 2.13), since data were nonparametric due to uneven sample size 2.7 Benzylaminopurine (BAP) treatment Buds were treated with BAP to observe whether a cytokinin c ould elicit the cell fate c han ge of epidermal cells similar to that which occurs in response to exogenous brassinosteroid (Clore et al., in revision) In order to selectively apply the hormone to the
! 8 : abaxial surface of the carpels, a 200 mg/ kg BAP lanolin mixture was made ( consistent w ith previous studies involving BAP treatment of tissues ) (Mosjidis et al., 1993; Wakushima, 2004). A BAP stock solution in 95% ethanol was thoroughly mixed into lanolin heated to 60 ¡ C. T he mixture was maintained at 60 ¡ C for 1 h to allow the ethan ol to co mpletely evaporate. C ontrol lanolin paste (with ethanol but minus BAP) was made using the same method. To treat the buds with BAP or control lanolin, a window was first cut into the bud to expose a fusing stage carpel on an intact plant or stem cutting. The lanolin mixture was then applied to the abaxial surface of the carpels with a wooden applicator stick, tapered at the tip. All manipulations took place under a dissecting microscope. Each bud was immediately covered loosely with a plastic bag secured w ith tape to prevent loss of moisture. I ntact plants or stem cutting s were incubated for 72 h in the growth chamber (25 ¡ C, 16 h/8 h light/ dark photoperiod). T reated buds were subsequently excised and fixed for light microscopy (see section s 2.3 and 2.4 ). 2. 8 DNA isolation for use in PCR Alkaloids and flavonoids produced in C. roseus may interfere with the isolation of DNA (St Pierre et al., 1999; Siddiqui et al., 2011). Therefore, genom ic DNA was isolated using Ultra Clean $ Soil DNA Isolation Kit (MoBio Labo ratories, Inc. Carlsbad, CA, USA ), which we reasoned would be useful for removing such contaminants. Approximately 0.15 g of sepals were removed from both fusing and post fusion buds pooled and frozen using liquid nitrogen. T issue was thawed before isola tion, which was
! 8; conducted as per the manufacture 's instructions. Isolated DNA product was analyzed on a 1.2% (w/v) agarose gel containing ethidium bromide following electrophoresis. 2.9 Polymerase Chain Reaction (PCR) PCR was used to amplify the BRI1 g ene from genomic DNA using degenerate primers ( dBR2, forward 5' GCNGARATTGGARACNATHGGNAARATHAARCA 3' and dBR5, reverse 5' GCCATNCCRAARTCNSWNACNCKNGCMTC 3') designed based upon the BRI1 amino acid sequence from Arabidopsis thaliana and ordered from Integrat ed DNA Technologies ( Montoya et al., 2002). Ten microliters ( 10 l ) of Taq mastermix (Qiagen HotstarTaq $ Plus Master Mix Kit), 1 l forward and reverse primers (100 pm/ l) 6 l H 2 O, and 2 l of template were used f or a final 20 l reaction volume Two du plicate DNA samples and a negative control with no template were placed in the thermocycler under the following conditions: 5 min at 95 ¡C, then 30 cycles of 94 ¡C, 45 sec at 55 ¡C, and 90 sec at 72 ¡C, all followed by a final 10 min extension at 72 ¡C. PC R products were analyzed using a 1% (w/v) agarose gel containing ethidium bromide following electrophoresis 2.10 Cuticle permeability assays Buds containing various stages of carpels w ere dissected to remove the outer whorl organs Carpels with attached receptacle were inverted into approximately 300 l of 0.05% (w/v) TBO (toluidine blue O ) in PBS, pH 7.4 or 0.1% or 0.05% (w/v) ruthenium red in 50 nm PBS in a 96 well plate for 2 min. C arpels and apical end of the receptacle were dyed. They were then brie fly rinsed in approximately 300 l water in a
! 8< 96 well plate. The dyed carpels were photographed and measured (see section 2.12). See figure legends 3.6 and 3.7 for numbers of replicates 2.11 Chlorophyll leaching assay S epals, petals, and anthers were rem oved from b uds cont aining post fusion carpels Five sets of postfusion or fusing carpels and their attached receptacles were inverted into 30 l of 80% EtOH along the sides of a 96 well plate, avoiding contact of the receptacles with ethanol (Fig. 2.1). One well was prepared for each timepoint (30, 60, 90,120 and 150 minutes). To minimize light exposure and evaporation, the plate was covered with a PCR plate seal cover (3M) and foil. After the appropriate time points, 5l were removed and added to 45 l 80% (v/v) EtOH in a new 96 well plate and absorbances read at 647 nm and 664 nm using a Bio Tek % Synergy HT multidetection microplate reader. Figure 2.1 Diagram illustrating five carpels inverted into a 96 well plate into ethanol used for the chlorophyll leaching assay, where R = receptacle, C = carpel, and E = ethanol.
! 8= 2.12 Dissecting and compound microscope imaging and digital image proces sing Gynoecia were visualized using a dissecting microscope, while tissue sections were observed under a compound microscope. All pictures were taken with a Canon Digital Powershot G3 Camera with an attached Max V iew Plus Ocular Adaptor¨ (ScopeT ronix). Bri ghtness and contrast were adjusted and microscope figures constructed using Adobe Photo shop $ version CS5 (Adobe Systems Inc., San Jose, California, USA ). The data resulting from these assays were not normally distributed as determined by the Anderson Darli ng coefficient, thus the Mann Whitney U Test was used as opposed to a parametric T test (see statistics section 2.13). 2.13 Statistics SAS $ (version 9.2) was used to calculate standard error and conduct Mann Whitney U Tests (alpha = 0.05) f or the wheat l eaf unrolling bioassay and chlorophyll leaching assay (SAS Institute Cary, NC, USA; results combined with that of M. Tatum). At least 3 replicates per time point were used to calculate the mean absorbances in the chlorophyll leaching assays, and graphs wer e made using Microsoft Excel (version 12.1.2)
! 8> Chapter 3: Results 3.1 Treatment with Brz2001 interferes with normal carpel development Histological sections were made and investigated to gain insight into the morphological defects observed during previous experiments using brassinosteroid inhibitors, spironolactone and Brz2001 (Kahn, 2006 New College thesis; observations of M. Tatum in Clore et al., in revision). Former student, M. Tatum sectioned 7 buds treated in the 5 15 M range of Brz2001 and one 1 M bud and tentatively concluded that 5 M concentrations or greater inhibited fusion. To verify and extend her results I sectioned 1, 3, and 5 M samples (one each) along with 4 DMSO (solvent for Brz2001) controls. Results were consistent with th ose of M. Tatum in that in both data sets, cross and longitudinal sections of Brz2001 treated gynoecia displayed inco mplete cell dedifferentiaton along the fusion plane when concentrations at or above 5 M were used (Fig 3.1) L ower conc entrations (or DMS O alone) resulted in complete dedifferentiation (Fig. 3.2)
! 8? Fig. 3.1 Evidence for fusion defects in carpels that developed in the presence of higher concentrations of Brz2001. a) Longitudinal section of a 10 M Brz2001 treated postfusion stage gynoecium before significant style elongation. Fusion does not appear complete as indicated by openings along the fusion plane (arrows). The cells surrounding these spaces display epidermal like characteristics. b) Longitudinal section of a DMSO treated control gyn oecium also in the postfusion stage exhibits normal fusion. c) Cross section of the stigmatic region of a postfusion stage gynoecium treated with 5 M Brz2001 indicates incomplete fusion. Many of the cells along the fusion plane (arrowhead) have not dediff erentiated, especially near the center. d) Cross section through the stigma of a postfusion DMSO treated control gynoecium reveals normal fusion in which the fusion plane is less distinct. Results representative of a total of 8 samples treated with 5 15 M Brz2001 (a & c) and 4 samples treated with DMSO (b & d) (d ata combined with that of M. Tatum, b d sectioned by author). Bars in a b = 200 M and c d = 100 M.
! 9@ Fig. 3.2 Cross section of a 3 M Brz2001 treated bud displays normal carpel fusion. Carpels s uch as these, treated with concentrations of less than 5 M Brz2001, demonstrated no perturbations in fusion like those seen in higher concentrations (as in Fig. 3.1). Results representative of 3 samples treated with 1 3 M Brz2001. Bar = 15 M. More sp ecifically, in the samples treated with high inhibitor concentrations, normally dedifferentiating cells remained small and cuboidal and the fusion plane was more distinct in treated specimens (Fig. 3.1a, c) as compared to the DMSO treated controls (Fig. 3 .1b, d) which appeared normally fused. Longitudinal sections of buds treated with high concentrations of Brz2001 revealed openings along the fusion plane intermittent with areas of dedifferentiated cells (Fig. 3.1a). In contrast, a control of comparable a ge displayed homogenous parenchyma tissue with no apparent suture line (Fig. 3.1b). Likewise, a cross section through a 5 M Brz2001 treated bud revealed a discernable fusion plane with little apparent dedifferentiation (Fig. 3.1c) whereas the
! 9" controls no longer had a dist inct fusion plane (Figs. 3.1 d). These effects were seen consistently in concentrations of 5 M and greater. Below 5 M, the fusion plane in the gynoecia appeared normal (Fig. 3.2 ). This observation was consistent with the fact that these gynoecia provided resistance to manipulation with a needle unlike the gynoecium depicted in figure 1.7c of the Introduction (Clore et al., in revison) which had been treated with brassinosteroid inhibitor 3.2 Treatment with both epi BL and Brz2001 resul ts in normal gynoecial development I tested whether treatment with exogenous brassinosteroid could rescue the effects of buds treated with a brassinosteroid inhibitor by treating stem cuttings with epi BL and Brz2001. C ontrol s were conducted to assess the specificity of the inhibitor. Dual treated fusing and posfusion stage carpels were analyzed for changes in morphology. The combination of epi BL and Brz2001 appeared to interfere with rate of development and was lethal in many of the cuttings, which was no t the case for treatment with the epi BL (or spironolactione) alone (Clore et al., in revision ; Kahn, 2006 ) The growth rate slowed considerably with use of both compounds. Leaves began to brown at the tips after a few days in scintillation vials, and buds produced progressively sma ller flowers (not shown). O ther potential causes were ruled out such as the water source (the results were the same whether dI or tap water was used), solvent effects (solvent alone did not have these negative effects), and where stem cuttings were maintained (growth chamber or greenhouse). At least 3 replicates of each stage were probed; however, only 4 fully mature gynoecia that developed in the presence of both compounds could be examined due to slow development of the carpels (Table 3.1).
! 98 Table 3.1 Assessment of fusion following treatment with epi BL and Brz2001 Number of gynoecia probed for fusion defects _____________________________________________ ______________________________ Stage in development Trial 1 Trial 2 Trial 3 Trial 4 Total Normal Total Abnormal Late fusion 3 1 1 5 Early postfusion 4 3 7 Postfusion 1 3 4 Late postfusion 1 1 Late post w/ style elongation 2 1* 1 3 1 indicates abnormal fusion Nonetheless, examine d buds demonstrat ed normal ca rpel development, i.e. fusion in all but one specimen Buds responded as follows to a dissecting needle lowered between the carpels. Fusing stage carpels could be separated completely but with some resistance whereas postfusion carpels were essentially des troyed (i.e. they were severely damaged ) since the deeper tissu e appeared to be solidly fused One fully mature gynoecium with some style elongation exhibited an abnormally distinct suture line and was easily separated. Its corolla exhibited some browning indicating possible necrosis so it could therefore be considered an outlier. The remaining responses to dissecting needle manipulation were consistent with those obtained using untreated carpels (Kahn, 2006; Clore et al., in revision).
! 99 3.3 Wheat leaf u nrolling bioassay suggests brassi nosteroid like activity of carpel exudates Carpel exudates (collected from solidified medium that had previously been in contact with 5 carpel pairs) were tested for brassinosteroid like activity using a wheat leaf unrolli ng bioassay ( Wada et al., 1984; Fig. 3.3). Percent of etiolated wheat leaf unrolling from the exudates was compared to that of the brassinosteroid, epi BL. B ecause ethanol ( epi BL solvent ) appeared to have a slight unrol ling effect, carpel exudates were te sted in the presence of water and ethanol. The data were combined with replicates performed by M. Tatum, justified by the Mann Whitney tests (alpha = 0.05) that demonstrated the percent unrolling of controls performed by each experimenter did not differ si gnificantly (ethanol control, p=0.1209; water control, p= 0.1221). Each treatment (carpel exudates, ca rpel exudates in ethanol, and 2 4 epibrassinolide) was shown to promote significantly greater unrolling than its a ppropriate solvent control (H 2 O or EtOH) using the Mann Whitney U Test (alpha = 0.05; Fig. 3.3). The mean percent unrolling of carpel exudates in the presence of water alone was slightly lower than the mean percent unrolling of exudates obtained with ethan ol. C arpel exudates in ethanol had a comp arable percent of unrolling to that caused by treatment with 0.01 g/mL epi BL (Fig. 3.3).
! 9: Fig. 3.3 Percent unrolling of etiol ated wheat leaves by epi BL, carpel exudates, carpel exudates in the presence of EtOH, EtOH control, and H 2 O control are shown (combined data with that from M. Tatum). Asterisks indicate significant differences in percent unrolling from their respective controls, E tOH (solvent for epi BL) and H 2 O as determined by the Mann Whitney U Test (alpha = 0.05). Carpel exudates stimulated significant unrolling of etiolated wheat leaves (p=0.004 and p=0. 0055, for carpel exudates in H 2 O and EtOH respectively). A concentration of 0.01 g/mL 24 epibrassinolide promoted unrolling, as well (p=0.0002). Since EtOH (solvent for epi BL) seemed to ha ve an effect on unrolling, carpel exudates were also tested in the presence of EtOH. Bars indicate standard error of the mean. N = 17 for BR, 11 for carpel, 6 for carpel+EtOH, 22 for EtOH, an d 18 for H 2 O 3.4 BAP does not elicit cell fate changes in a baxial cells of the carpels Treatment with the cytokinin, BAP was used to test whether another plant hormone could elicit cell fate change in abaxial epidermal cells as does exogenous brassinosteroid. Both longitudinal (not shown) a nd cross sections wer e analyzed. Tests revealed that b uds treated with BAP did not ex hibit changes in cell fate like that induced by e xogenous brassinosteroid (Fig. 3.4 ). The cells appeared uniformly epidermal (Fig. 3.4a) except when the carpel was obviously wounded upon appl ication of the lanolin paste (data not shown) Although the lanolin used to apply the hormone was not easily identifiable after fixation and extensive rinsing (although sometimes a remnant/darkened area could still be seen on the carpel surface as seen in Fig. 3.4a) it was verified to be
! 9; present after the initial incubation with the hormone. Brassinosteroid treated samples (Kahn, 200 6) are included for comparison. Fig. 3.4 Results of BAP treatment of carpels compared with previous experiments using trea tment with epi BL on carpel abaxial surfaces. a) BAP treated fusing stage carpel displayed a normal epidermal layer even on the site of application, which could be discerned by a darkened area at the carpel surface (arrow). b ) Prefusion carpel treated with epi BL displayed frequent oblique and periclinal divisions on the site of application, whereas the cells on the untreated side (bottom) remained epidermal in fate. c) Fusing stage carpel treated with epi BL again demonstrating a layer of dedifferentiated cells like that elicited by carpel exudates. Bars in a = 15 M and b c = 10 M. 3.5 A PCR product of predicted size amplified from C. roseus DNA using degenerate primers for BRI1 Since littl e information is available on C. roseus brassinosteroid related genes I attempted to amplify the C. roseus orthologue of BRI1, which encodes the brassinosteroid receptor (Li and Chory, 1997). PCR was performed on C. roseus genomic DNA using degenerate primers for the BRI1 sequence designed based on conserved amino aci d residues in Arabidopsis (Montoya et al., 2002) The predicted size for BRI1 was ~318bp (Montoya et al., 2002). A distinct band was obtained between 300 400 bp in size (Fi g. 3.5 ), consistent with the predicted size. The replicate bands were measured to be
! 9< approximately 330 bp using the Duggleby DNA Size Mapper ( http://www.pangloss.com/seidel/Protocols/webmap.html ), which is very close to the predicted size The slight disparity may be due to error in the distance measurements used for the estimates or to a slight length difference between BRI1 in this species versus in Arabidops is A faint low molecular weight band seen in all three lanes, including the negative control, was likely the primers. Fig. 3.5 Visualization of C. roseus PC R product amplified using degenerate primers for BRI1 on an ethidium bromide stained agarose gel (after 30 cycles of PCR), where lanes b and c are duplicates. b c) The size of the product is between 300 400 kb, which is consistent with the predicted size o f the BRI1 sequence homologous to Arabidopsis d) Negative control exhibits a faint band (seen in all lanes) toward the bottom that is likely the primers.
! 9= 3.6 Cuticle p ermeability assays demonstrate distinct pattern s of staining that change throughout gyn oecial development Since cuticle modification may aid in the diffusion of the factor' and/or plays a role in the fusion process itself ( Verbeke, 1992a ; Lolle and Pruitt, 1999) changes in cuticle permeability were investigated in carpels at varying stages of development. Toluidine blue O (TBO) stains components of the cell wall under the cu ticle and therefore, the extent of staining correlates with the extent of cuticle permeability (Tanaka et al., 2004; Viosin et al., 2009). Carpels treated with TBO showe d distinct and reproducible pattern s of staining throughout development (Fig. 3.6 ). Prefusion stage carpels not yet in contact with each other display ed n o discernable staining (Fig. 3.6 a). Upon contact but prior to the fusing stage staining was seen on the adaxial surfaces near t he base of the carpels (Fig. 3.6 b). Fusing carpels stained circumferentially on the adaxial and abaxial surfaces in the region that later de velops into the stigma (Fig. 3.6 c). The tops of the carpels remained unstained in areas where the carpels were not in contact. In addition the base of the carpels were unstained; however, staining appeared to dip downward into the region of the future style in a V formation where the carpels were in contact. Postfusion carpels exhibited well stained stigmatic tissue, except in the very distal tip regions (Fig. 3.6 d, e). After the style had begun to elongate, the tip remains relatively unstained while the stigma and developing skirt were stained (Fig. 3.6 e, arrow). Ruthenium red ( Curvers et al ., 2010 ) also stains a component of the cell wall (pectins) and produced very similar albeit fai nter staining patterns (Fig. 3.7a e ). Results of staining using 0.1% and 0.05% ruthenium red were pooled, since n o differences were observed between concentrati ons.
! 9> Figure 3.6 Toluidine blue O stained carpels demonstrate dis tinct pattern s during development. a) Prefusion carpels have a small but noticeable space between them and are unstained. b) Prefusion carpels have just come in contact and exhibit slight staining on the base of the carpels on the adaxial (inner) surfaces (inset). c) Fusing carpels show staining circumferentially on both the adaxial and abaxial surfaces (a rrowhead, inset). d) Early postfusion carpels prior to style elongation are well stained, except for the distal portions of the future stigmatic region (a rrowhead, inset). e) A mature gynoecium with some style elongation demonstrates circumferential staining in the stigma and d eveloping skirt (arrow), but much less in the distal tips. Results were reproducible and images representative of a minimum of 5 dis sections per stage depicted. Bar = 200 m
! 9? Figure 3.7 Ruthenium red stained carpels show the same basic stain ing pattern as TBO. a) Prefusion carpels have a small but noticeable space between them and are unstained. b) Prefusion carpels have just come in contact and exhibit slight staining on the base of the carpels on the adaxial surfaces (inset). c) Fusing carpels show staining circumferentially on both the adaxial and abaxial surf aces (inset). d) Early post fusion carpels prior to style elongation are we ll stained, except for the distal portions of the future stigmatic region e) A mature gynoecium with some style elongation demonstrates circumferential staining in the stigma and developing skirt (arrow), but not in the distal tips. Results representative of a minimum of 3 dissections per stage depicted. Bar = 200 m
! :@ 3.7 Results of attempts to adapt a chlorophyll leaching assay for assessing changes in cuticle p ermeability during carpel development In conjunction with the cuticle permeability assays, a chlorophyll leaching assay was modified for exploring c hanges in cuticle permeability throughout development (Lolle et a., 1997; Viosin et al., 2009). Fusing a nd postfusing carpels were used. P refusion carpels were not used due to their tiny size and difficulty of manipulat ion. Preliminary data indicate differ ence s in rate s of chlorophyll diffusion between fusing and po stfusion stage carpels (Figs. 3.8 and 3.9 ). Postfusion carpels were tested first since they are larger and easier to manipu late. R aw absorbance values at 647 nm and 664 nm increased over time for the postfusion carpel trials indicating diffusion of both chlorophyll a and chlorophyll b Although fusing stage carpel trials did not exhibit as steep an increase in absorbance values (slope about 10x lower), the initial 664 nm values were significantly greater than for postfusion (Mann Whitney U Test, p=0.0463, alpha=.05).
! :" Figure 3.8 The raw absorbance values from the chlorophyll leaching assay using postfusion stage carpels. Means from 3 replicates for 30, 60, and 90 minutes and 2 replicates for 12 0 minutes are shown. Bars indicate + standard error (664nm) and standard error (647nm).
! :8 Figure 3.9 The raw absorbance values from the chlorophyll leaching assay using fusing stage carpels. Means from 3 replicates for 30, 60, 120, and 150 minutes and 6 replicates for 90 minutes are shown. Bars indicate + standard error (664nm) and standard error (647nm).
! :9 Chapter 4: Discussion This thesis project investigated the nature of the transmissible factor' involved in C. roseus cellular dedifferentiat ion during carpel fusion. It also tested the hypothesis that changes in cuticle permeability take part in this process. Previous studies suggest that a necessary factor' is exchanged between carpels during fusion (Verbeke and Walker, 1986; Siegel and Verb eke, 1989) and is neither one of the "classic" plant hormones (cytokinin, auxin, or gibberellic acid ; personal communication from J.A. Verbeke to A.M.Clore ) nor is proteinaceous in nature (Kahn, 2006; Clore et al., in revis ion). Further research suggests that the recently identified plant hormone, brassinosteroid, may be involved in this process (Kahn, 2006; Clore et al., in revision). Several experiments in the present study further addressed the hypothesis that brassinosteroid signalin g mediates deeper t issue fusion, and also the changes in cuticle permeability occur during carpel development, since cuticle modification had been previously suggested to occur during fusion (Walker, 1975c; Lolle et al., 1997) but not yet explicitly tested Experiments in the present study support the hypothesis that brassinosteroid signaling is important for C. roseus carpel fusion. First, buds treated with the brassinosteroid inhibitor, Brz2001, demonstrated histological perturbations in fusion. The histological sections, when combined with those of M. Tatum demonstrated inhibited cellular dedifferentiation in buds treated with 5 M or higher concentrations of the inhibitor but not in buds treated with lower concentrations or DMSO alone. At or above 5 M, c ross sections re vealed a more distinct fusion plane in which many normally dedifferentiating cells rem ained epidermal, particularly toward the center of the tissue. Similarly, longitudinal sections exhibited disruptions along the fusion plane where cells
! :: did not dediffere nt iate, indicating some disruption in fusion. These results are consistent with the morphological assessment of carpels in buds treated with both Brz2001 and the older, less specific brassinosteroid inhibitor, spironolactone, in which, the carpels were eas ily separable when probed with dissecting needle (Kahn, 2006; observations of M. Tatum in Clore et al., in revision). In addition, in the present study, exogenous application of the brassinosteroid, 24 epibrassinolide, was shown to rescue the inhibitory ef fects of Brz2001 alone, even though the combination of the compounds had detrimental effects on overall d evelopment. All but one gynoecium that developed in the presence of both compounds displayed normal fusion upon probing s, consistent with the notion th at Brz2001 specifically inhibits brassinosteroid synthesis (Sekimata et al., 2001) The morphological and histological results in dicated that deeper tissue fus ion is dependent on brassinosteroid like activity. Superficial adhesion may occur by a different pathway since the inhibitors do not prevent the two carpels from lightly adhering to one another (Kahn, 2006; observations of M. Tatum in Clore et al., in revision). T he results of the wheat leaf unrolling assay also suggest a role for brassinosteroid sign aling in carpel fusion. Carpel exudates exhibited brassinosteroid like activity in etiolated wheat leaves and stimulated significantly greater un rolling than the controls. M ean percent unrolling promoted by carpel exudates in the presence of ethanol were c omparable to the effects of exogenous brassinosteroid. A dditional replicates of the wheat leaf unrolling assay using carpel exudates in the pres ence of ethanol would be useful; however, considering th is condition had the fewest replicates due to a fungus p roblem that started to grow on the wheat when subsequent replications were attempted (but not used for analysis) Cytokinins (moderately) and gibberellins (only slightly) do
! :; promote some unrolling in etiolated wheat leaves but not with t he efficacy of bras sinosteroid. A uxins inhibit wheat leaf unrolling (Wada et al., 1985). It could stand to reason that gibbe rellins or cytokinins may have contributed to the unrolling in the present study; however, the collective results implicate brassinosteroid as a major morphogen in the fusion process. Furthermore, a p plication of the cytokinin BAP on the abaxial surfaces of the carpels did not result in cellular dedifferentiation. It was hypothesized that if the cytokinin were to have any effect, it would result in small newly divided cells (since it is known to stimulate cell division in a number of plant tissues; Werner et al., 2001) rather than layers of dedifferentiated cells like those triggered by epi BL. Ex ogenous application of epibrassinolide elicits epidermal ce ll dedifferentiation, simila r to that seen with the actual factor' (Durbak, 2004; Clore et al., in revision). In contrast, BAP treated carpels displayed uniformly epidermal tissue on the site of application, identified by anticlinally dividing cells that were not isodiametric, but rather, more tabular in nature It is interesting to note that in some samples very occ asional anticlinal divisions could be seen a few cells away from the fusion plane (not shown) ; however, this is not uncommon in untreated buds Nonetheless, epidermal cells treated with cytokinin (Fig. 3.4a) do not resemble those treated with factor' (Fig. 1.4b), as do those treated with epi BL (Fig. 3.4b, c). Additional replicates of BAP treated buds would be advantageo us, since only two repli cates f r om each plane of section were treated and examined E fforts to characterize the signaling pathway on a mo lecular level were explored, and amplification of the BRI1 gene in C. roseus was attempted. A C. roseus DNA PCR product of the predicted size for the BRI1 gene was amplified using degenerate primers.
! :< Distinct b ands of approximately 330 bp were obtained in the replicates comparabl e to that of the predicted size of ~318 bp (measured using Duggleby DNA Size Mapper; Montoya et al., 2002) in Arabidop sis while the negative control did not exhibi t a band in this range. Sequencing of the obtained product is necessary to make any definitive conclusions, but could propel interesting future research (as discussed in future directions). Considerable eviden ce indicates that brassinosteroid signaling is involved in cellular dedifferentiation during carpel fusion. However, superficial fusion still occurs in the presence of brassinosteroid inhibitors. Even at h igh Brz2001concentrations, carpels were able to sup erficially adhere (Kahn, 2006; Clore et al., in revision), consistent with the hypothesis that superficial adhesion and deeper tissue fusion occur by two independent pathways (Clore et al., in revision) Additional signaling pathways; therefore, may be nec essary to facilitate fusion. Cell wall material, deposited upon contact of carpel adaxial epidermal cells, is thought to promote adhesivity between the carpels (Walker, 1975b). Characterization of these new materials may provide insight into the superficia l adhesion process during carpel fusion. Cuticle modification has also been suggested to occur during gynoecial development and may conceivably help to facilitate passage of signaling molecules involved in carpel fusion (Walker, 1975b; Lolle et al., 1999) Cuticle permeability assays using toluidine blue O and ruthenium red indicated distinct and reproducible patterns of cuticle permeability changes throughout development. In early prefusion carpels the cuticle appears to be highly impermeable, since the c arpels did not take up stain initially, but then light staining was observed just after contact but prior to dedifferentiation. It is
! := unclear what prompts changes in permeability sheer physical contact between carpels, or perhaps brassinosteroid signaling. It should be noted, though that physical contact appeared to be necessary for changes in cuticle permeability to occur, as evidenced by consistent lack of staining in carpels just prior to contact. Results of experiments in which prefusion carpel exudate s were applied to recipient adaxial epidermal cells and elicited some morphological changes in cell walls but not dedifferentiation (Durbak, 2004; Clore et al., in revision), suggest that these very early changes in cuticle permeability may precede the bra ssinosteroid pathway. During later stages of fusion the cuticle consistently becomes more permeable circumferentially (on adaxial and abaxial surfaces) except at the distal tip portions where epidermal contact does not occur. Increased permeability on th e adaxial and abaxi al carpel surfaces is logical, since the abaxial surfaces also have the potential to dedifferentiate (Siegal and Verbeke, 1989; Kahn, 2006; Clore et al., in revision). The observed changes in cuticle permeability during C. roseus carpel fusion may contribute to our overal l knowledge of organ fusion, although the full significance has yet to be determined. Following fusion as the gynoecium matures further, the stigma and skirt continued to stain circumferentially indicating increased perm eability, while the distal tips remained green (relatively unstained). Maintenance of i ncreased permeability for so long after fusion may have potential benefits in the reproductive success of the organism, particularly in pollen hydration for which incre ased cuticle permeability appears necessary (Lolle et al., 1997; Lolle and Pruitt, 1999). The basal portion of the stigma in C. roseus has been suggested to be the specific region that is receptive to pollen, which is compatible with its high level of perm eability (Kulkarni et al., 2001). Although cuticle
! :> permeability may be increased prior to brassinosteroid signaling, the somewhat hydrophobic nature of brassinosteroid may still be applicable since remnants of the cuticle have been observed all the way up to the postfusion stage (Walker, 1975c). Finally, a chlorophyll leaching assay was adapted for potential use in further investigating cuticle permeability during development Preliminary evidence does suggest a difference in chlorophyll leaching rate s in fusing and postfusion carpels indicating that the assay may have utility However, of greater interest are the differences in chlo r ophyll leaching rates between prefusion and fusing carpels since the initial onset of permeability changes occur during this time. Because the desirable stage carpels are very small, they are difficult to manipulate particularly down along the sides of the microplate well without dropping the entire receptacle into the well while attempting to avoid ethanol contact with the rec eptacle The young tissue is also very soft and easily damaged In addition the size of the carpels and their relatively pale color may mean the appreciable levels of chlorophyll cannot be leached from the tissue. It is unclear how to make this assay work successfully, espec ially in prefusion carpels. The assay is laborious to conduct and evaporation of the small volumes of ethanol would be difficult to prevent 4.1 Future d irections This study presented evidence of brassinosteroid involvement in C. roseu s carpel fusion. The ability of 24 epibrassinolide to rescue the affects of Brz2001 on fusion was demonstrated despite the fact that the combina tion of compounds led to stem cutting necrosis The results supported the idea that this compound specifically i nhibits brassinosteroid synthesis (Sekimata et al., 2001). A supplementary rescue experiment
! :? using direct application of the compounds onto the abaxial surfaces of the carpels, presumably with lanolin or agar (analogous to trea tment with BAP and exogenous epi BL alone) could be us ed to verify the results. Perhaps exo genous application could bypass the compromising health effects seen with incubation of the whole cuttings. In order to further investigate brassinosteroid signaling in the fusion process, addi tional analysis of the obtained PCR product should be performed To sequence the putative BRI1 ortholog for C. roseus, a prep arative gel sh ould be made and loaded with large amounts of PCR product and the resulting band excised, and then the DNA eluted and sent off for sequencing Once the BRI1 ortholog is sequenced it may be possible to visualize its expression patterns in the developing carpels by in situ hybridization (ISH). Traditionally, t o make hybridization probes for the BRI1 sequence, cDNA must be made. However, it is extremely tedious to isolate enough mRNA from such minute amounts of tissue. Previous attempts to isolate total RNA and subsequent cDNA production for each stage of development required 100 prefusion, 75 fusing and 50 postfusion carp el pairs, each excised from buds from about one to a few millimeters tall (Durbak, 2004). GeneDetect $ designs and makes labeled oligonucleotide pr obes based simply upon sequence; however these probes are not currently available for purchase. Provided such probes again become available characterization of BRI1 gene expression throughout gynoecial development may give insight into brassin osteroid signaling in carpel fusion. Eventually, ISH could be done for several of the components in the brass inosteroid p athway providing additional information on the potential signaling pathway during carpel fusion. Real time reverse transcriptase PCR may also be useful for quantification
! ;@ of BRI1 and related transcripts over a developmental time course if sufficient RNA co uld be isolated to make the required cDNA. The factor' has thus far not been directly analyzed largely due to the carpels' tiny size and the inability to obtain appreciable amounts of exudates. Solid phase microextraction (SPME) fiber s, relatively new t ools (Jain and Verma, 2011) may be sensitive and small enough to extract the factor' from between the very small individual carpels and could be used in gas chromatography mass spectrometry (GC MS) for subsequent identification of carpel exudates. SPME is a sample preparation technique to obtain analy tes in either liquid or gas phase and has successfully been used to analyze trace amounts of anabolic steroids in pig urine (Zhang et al., 2009). T his may be feasible since we have access to such a techni que at New College of Florida. The idea that there are multiple components to the factor' has been put forth (Verbeke, 1992a). The fact that even at high brassinosteroid inhibitor concentrations, some isolated cells manage to dedifferentiate (Fig 3.1a, c, p articularly in the peripheral portions of the fusion plane) may indicate incomplete inhibition of the brassinosteroid pathway, or it may indicate the presence of a complementary or partially redundant additional pathway. Chemical characterization of the f actor' may shed light on this possibility. Anothe r future aspect of this study would be to determine modifications in cuticle and cell wall materials. In the present study it was demonstrated that changes in cuticle permeability occur throughout develop men t, but it is mysterious how and precisely when these changes occur. A more thorough idea of the timing could be obtained using the chlorophyll leaching assay to compare prefusion and fusing carpels (if the young prefusion carpels can be manipulated). Howev e r, more trouble shooting is necessary as
! ;" discussed earlier One possible alternative to measur e chlorophyll diffusion rate would be to capture diffused chlorophyll on a solid medium (perhaps containing ethanol) A solid medium would provide a more managea ble surface in which receptacle ethanol contact could be avoided, and would also diminish the evaporation problem. A direct investigation of differential cuticle composition throughout development would shed light on how these cuticle changes transpire at the chemical level One way to do this would be to compare the components of lipid extract s made from different stage carpels (Lolle et al., 1997; Lolle and Pruitt, 1999) although again the size of the carpels may be a limitation S ome research progres s has been made on C. roseus carpel fusion but there is certainly a great deal more to learn from this interesting phenomenon. Using a combination of techniques, such as the cuticle permeability assays and GC MS in conjunction with a molecular biology ap proach the signaling pathways and extracellular matrix modifications during gynocial development can be elucidated. Such knowledge could make contributions to the greater understanding of cell cell signaling, organ fus ion, and cell dedifferentiation.
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