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EXPLORING CHANGES IN SURFACE STRUCTURE AND POTENTIAL ROLES FOR REACTIVE OXYGEN SPECIES SIGNALING DURING POSGENITAL CARPEL FUSION IN CATHARANTHUS ROSEUS BY HELENA LILY BENEDICT A Thesis Submitted to the Division of Natural Sciences New College of Florida In partial fulfillment of the requirements for the degree Bachelor of Arts Under the sponsorship of Dr. Amy Clore Sarasota, Florida May 2013
ii A CKNOWLEDGEMENTS Many thanks to my committee members, Dr. Beulig, Dr. Clore, and Dr. McCord, for taking the time to read the thesis and to sit on my bacc committee. Thanks especially to Dr. Clore, for your incredible patience and wisdom in guiding me through this process. As a professor, advisor, thesis sponsor, and mentor you've been more than k ind and always helpful, and continually insipired me and encouraged my curiosity. I'm grateful to Mr. Joel Thurmond for all his assistance in the New College lab and to Mr. Ed Haller at the University of South Florida for helping me use the microscope ther e, as well as for answering questions and sharing resources. Thanks to Kaija Goodman for the patient demonstrations, and for keeping the plants alive. Thanks to my parents for their boundless love; you've given me more than I think you know. Thanks to Tesl in for keeping me sane and to Jed Walsh for late night reassurances. Thanks to my family and friends for all the support. And thank you Sam, for everything.
iii T ABLE OF C ONTENTS Acknowledgements ............................... ........ ... ................................................ .............. ..... ii Table of Contents ................................................................................... ..... .... ......... .......... iii List of Figures .................................. .................................................... ........ .... .................. iv Abstract ..................................................................................................... ... .... ... .... ............. v C hapter 1 Introduction ................................................... ..................... ................ ... .......... 1 1.1 Evolution and Anatomy of Female Reproductive Structures................ .. ......1 1.2 Organ Fusion in Catharanthus roseus .... .. ...... ................. ............................ .. 5 1.3 Evidence of a Role for Brassinosteroid Signaling 9 1.4 Overview of the Brassinosteroid Signaling Pathway .. 10 1.5 Brassinosteroids and Plant Development 13 1.6 A Case for a Role for the Cuticle 17 1.7 A Possible Role for R eactive Oxygen Species Signaling 1.8 RO S Signaling in Plant Development 1.9 Thesis Overview 31 Chapter 2 Materials and Methods 33 2.1 33 2.2 33 2.3 35 2.4 36 2.5 38 2.6 38 Chapter 3 40 Chapter 4 47 Chapter 4 5 5
iv L IST OF F IGURES AND T ABLES Figure 1.1 Diagram illustrating the parts of a flower 2 Figure 1.2 Schematic of types of organ fusion in plants 4 Figure 1.3 Catharanthus roseus blossom 5 Figure 1.4 Fine surface structures f rom a previous SEM study 7 Figure 1.5 Brassinosteroid signal transduction pathway 11 Figure 1.6 Structure of the plant cell cuticle 18 Figure 2.1 Dissection of dried bud to reveal prefusion carpels 37 Figure 3.1 Comparison of postfusion carpels from bu ds treated with Brz2001 and DPI 41 Figure 3.2 Higher magnification of the fusion suture in gynoecia exposed to Brz2001 and DPI 42 Figure 3.3 Fine surface structures of prefusion carpels 43 Figure 3.4 DAB staining for hydrogen peroxide over the course of carpel fusion 44 Table 3.1 Summary of effects of different treatments on the fusion process 46
v EXPLORING CHANGES IN SURFACE STRUCTURE AND POTENTIAL ROLES FOR REACTIVE OXYGEN SPECIES SIGNALING DURING POSTGENITAL CA RPEL FUSION IN CATHARANTHUS ROSEUS Helena Lily Benedict New College of Florida, 2013 ABSTRACT During normal development, the carpels of the Madagascar periwinkle Catharanthus roseus L. arise as two distinct organs but later make contact and fuse t o form a single gynoecium. Significantly, approximately 400 epidermal cells along the site of fusion change their pl ane of division and eventually r edifferentiate into less specialized parenchyma tissue. Understanding the mechanisms by which neighboring ce lls communicate and orch estrate developmental processes is important to the study of plant development making this a potentially useful model system. P reviously documented increase s in carpel cuticle permeability as fusion progresses, together with liter ature reporting altered cuticle properties in Arabidopsis mutants expressing abnormal organ fusion, suggest that changes in cuticle structure may play a role in C. roseus carpel fusion. Recent research also demonstrates that brassinosteroid signals exchang ed between prefusion carpels play an important regulatory role in the fusion process, as inhibiting brassinosteroid biosynthesis results i n abnormal, superficial fusion. The present study employed scanning electron microscopy to chart changes in surface st ructure of both normally fusing carpels and those that were treated
vi with the brassinosteroid biosynthesis inhibitor Brz2001 throughout development. Attempts were also made to further elucidate the carpel fusion signaling pathway through the use of diamino benzidine tetrahydrochloride ( DAB ) staining and the inhibition of NADPH oxidases two tools commonly used to investigate potential roles of reactive oxygen species. The results suggest that hydrogen peroxide signaling merits future investigation as a likel y player in the fusion process. Furthermore, the SEM work generally corroborated earlier studies of gynoecial ontogeny of extracellular structures that may be impor tant in the fusion, and supported the role of brassinosteroids in this fusion. Dr. Amy Clore Division of Natural Sciences
1 Chapter 1: Introduction The present study examines carpel fusion in the Madagascar periwinkle otherwise known as Catharanthus roseus (L.) G. Don Through this process, two organs that arise a s distinct entities grow together to form a single structure. The fusion involves a characteristic redifferentiation, wherein cells along the fusion plane lose their epidermal character and become indistinguishable from the surrounding parenchyma tissue. W hile the cellular and morphological changes that accompany C. roseus carpel fusion are reasonably well characterized, the nature of the signals exchanged between the developing carpels and the details of how those signals drive development are still being investigated. There is a possibility that changes in the fine structure of contacting surfaces play a role in facilitating fusion, similar to those that accompany pollen stigma interactions (Lolle, 1999). This study examines the relationship between some e stablished chemical signals and the fine surface structure of carpels at various stages of fusion, and investigates a potential role for r eactive oxygen species signals (which are known to influence tissue patterning in other systems) in coordinating the f usion process. A review of the rele vant literature, including what is currently known about Catharanthus roseus carpel fusion and what clues about the process can be gleaned from other systems, will be presented to provide context for the experimental work 1.1 Evolution and Anatomy of Female Reproductive Structures The gynoecium is the female reproductive organ of flowing plants (Figure 1.1). The structural unit of the gynoecium is known as the carpel, which consists of a stigma,
2 style, and ovary. The stig ma, at the distal end of the carpel, is where the pollen first makes contact; a pollen tube then grows down through the style and into the ovary, allowing the male gamete access to the ovules inside. This division of labor, between pollen reception at the stigma and housing of ovules in the ovary, was a major evolutionary step for angiosperms (Arber, 1907; Taylor and Hickey, 1996). Besides cushioning and protect ing the female gametes and thei r nutritive tissue, the carpel allows for greater flexibility in r eproductive structure by providing a dedicated surface for pollen capture the ovules themselves no longer need to be available and amenable to the pollen grains (Leslie and Boyce, 2012). Due in part to this increased plasticity, angiosperms (as compared to gymnosperms) have exhibit ed great diversity and increased complexity in both the structural and spatial arrangement of their reproductive organs, and in their interactions with pollinators (Leslie and Boyce, 2012). Interestingly, a recent hypothesis sug gests that the development of a closed carpel (angiospermy) was made Fig. 1.1: The parts of a flower. Note that the gynoecium is here referred to as the pistil, although the two terms are used interchangeably. It is composed of the stigma, style, and ovary, and houses the female gametes. The gynoecium may consist of a single carpel, multiple distinct carpels, or of carpels several fused into a single structure. ( Image: Col orado State University Extension)
3 possible by changes in the architecture and composition of pollen tube cell walls that enabled faster tube growth (Williams, 2012). Increase d pollen tube growth rate allowed for greater s patial separation (via the int ervening style) of the pollen from the ovule without sacrificing fertilization time. This separation enhances pollen competition and helps isolate the ovule from potential patho gens (Walsh, 1992; Wang, 2 011). F urthermore, incr eased flexibility in style length is suggested as a driving force in the diversification of female reproductive structures (Williams, 2012). The gynoecium in some species is comprised of separate carpels forming distinct chambers. This pattern, however, is mostly confine d to basal angiosperms. R oughly 83% of extant species exhibit a pattern of fused carpels known as syncarpy (Endress, 1982). Syncarpy appears to have arisen repea tedly throughout angiosperm evolution suggesti ng that it confers significant evolutionary advantage (Armbruster, 2002). The fusion of carpels offers several obvious defense benefits, such as increased mechanical strength of the gynoecium and the ability to dedicate more resources to the outer wall (Stebbins 1974). Furthermore, co mputational models comparing a po carpous and syncarpous modes of reproduction under various conditions have suggested that syncarpy generates advantages in both quantity and quality of offspring, likely arising from the selective pressure of enhanced pollen competition (Armbruster, 2002). The fusion of carpels is thus an important evolutionary event, as well as being vital to the life cycle of many modern day angiosperms. Organ union in plants (including that of carpels) occurs by one of two processes (as r eviewed by Verbeke, 1992 and Endress, 2006). Congenital fusion (Figure 1.2 A)
4 describes the genesis of compound structures that develop as one unit, with common meristematic origin (Endress, 2006). Postgenital fusion, by contrast, refers to structures wh ich arise as distinct organs and fuse only after significant independent development (Figure 1.2 B and C ) Fusion in this case depends on contact between the epidermal cells of the fusing organs (Verbeke, 1992.) Both modes of fusion occur frequently in gyn oecial development, although congenital fusion appears to be more widespread (Endress, 2006). Closure of the locule (the space within an individual carpel which houses the ovule) is an important process which, like carpel fusion, relies on the activity of epidermal cells at the carpel surface. Work in several families of basal angiosperms shows that carpel closure leading to true angiospermy arises through polysaccharide secretion, postgenital fusion of the walls of a single carpel or a combination of the two (Endress and Igersheim, 2002; Fig. 1.2: A diagram of types of organ fusion (see text for more details). Carpel fusion in C. roseus is an example of spatially restricted postgenital fusion (C), in which the fusion is restricted to a limited area and d ependent on direct contact of the fusing surfaces. (Adapted from Walker, 1975).
5 Sol tis, 2004; Endress, 2006). The most ancient families achieve a closed carpel by secreting polysaccharides which fill the gap between carpel walls and seal the ovules off from the outside (Lloyd and Wells, 1992). More a dvanced families rely on postgenital fusion of the carpel walls to form a continuous closed surface (Endress and Igersheim, 2002). This continuum of fusion types suggests that the competence of developed tissue to fuse was important in the evolution of new structures and modes of reproduction. 1.2 Organ Fusion in Catharanthus roseus Catharanthus roseus (L.) G. Don is a flowering plant endemic to Madagascar that is now enjoying widespread use in subtropical climates as an ornamental (Figure 1.3). It is a member of the family Apocynaceae (Dogbane family). In addition to producing cancer fighting alkaloids that have been used as pharmaceuticals (Van Der Heijden, 2004), C. roseus is notable in that it exhibits a unique form of postgenital carpel fusion tha t involves the redifferentiation of epidermal cells into less specialized parenchyma tiss ue (Walker, 1975a). This redifferentiation (sometimes termed dedifferentiation) is rare in plant development, as cells that have matured into specialized tissue throug h morphological and physiological changes do not often revert to a lesser specificity. Such C. roseus is in contrast to the gynoecial development exhibited by other species. Fusion proc eeds congenitally in Arabidopsis thali ana (Smyth, 1989; Verbeke, 1992). P ostgenital fusion therefore cannot be studied in Arabidopsis Fig. 1.3: C. roseus blossom (Image: Wikimedia Commons)
6 thaliana which include, among others, a fully sequenced genome (The Arabidopsis Genome Initiative, 2000) The unique cellul ar redifferentiation exhibited in C. roseus gynoecial development, together with the broader evolutionary significance of the fusion process, make s it an interesting topic of study. Of particular interest are the sign aling pathways that regulate fusion eve nts, since these are what ultimately determine the fate of the cells. First described in detail by Walker in 1975, the basic events of C. roseus carpel fusion are fairly well elucidated (Walker, 1975a,b; Walker, 1978; Verbeke, 1989). Over a time course of about nine hours, the two distinct carpels, which arise as side by side, initially horseshoe shaped primordia in the innermost whorl of the flower, make contact and fuse. Initial contact occurs when the carpels are about 260 m long. A t this stage, true f usion has not yet begun and buds at this point or earlier are known as prefusion (Walker, 1975a). H igh magnification scanning electron microscope studies of prefusion buds reveal that the adaxial surfaces which are fated to fuse are covered in a mesh lik e film with occasional larger, rope like fibrils ( Figure 1.4; Walker, 1975a). The identity and function of these films and fibrous structures remain open to speculation, although it was hypothesized that they might represent cell wall materials, such as c ellulose microfibrils, that extend into the cuticular layer (Walker, 1975b). By the time the carpels reach 350 m, fusion is well underway and previously epidermal cells along the contact zone have lost their epidermal character This is termed the fusion stage Contact zone cells, which have been dividing anticlinally (perpendicular to the organ surface) begin instead to undergo periclinal (parallel to the surface of the tissue), as well as oblique divisions (Walker, 1975b). These cells show changes in
7 Fig. 1.4: Sample scanning electron micrographs from a study by Walker (1975). The upper image depicts prefusion carpel s whose adaxial (inner) surfaces have not yet made contact, and arrows point to regions where a filmy covering can be seen peeling away from the body of the carpels. The lower two images were taken at a much higher magnification and illustrate the mesh lik e nature of this film as well as the presence of twisting rope like structures running across the adaxial surface of each carpel (Adapted from Walker, 1975b).
8 cytoplasmic organization, becoming highly vacuolated and richer in organelles such as golgi and rough ER (Verbeke, 1989). Significantly, fusion is contact mediated and spatially restricted: the distal tips of the carpels make contact, b ecome appressed, and fuse, while the basal most regions of the carpels remain unfused and free throughout development (Walker, 1975a). Once fusion is complete, the fused region continues to grow and develop as a single organ, maturing into the stigma, elon gated style, and distal portion of the ovaries (Boke, 1949). Unified growth and development is known as the postfusion stage and gynoecia are considered to be postfusion proper when they reach a height of about 550 m and the fusion plane is no longer app arent in cross section (Clore et al, in revision) The existence of a diffusible signaling factor exchanged between developing carpels was first hypothesized based on experiments using an impermeable barrier of gold foil. When placed between incipient car pels the barrier prevented fusion in areas where cell cell contact was prevent ed, while cells beyond the barrier continued to develop normally (Walker, 1978). Subsequently, it was shown that a porous, water permeable barrier did not impede fu sion (Verbeke and Walker, 1986), and that carpel secretions could be captured in agar impregnated polycarbonate barriers and used to induce a redifferentiation response when placed on the outer, non con t a c t ing abaxial surface (Siegal and Verbeke, 1992). Durbak (2004), G oodman (2012), and Clore (in revision) have shown that blocks of agar solidified growth medium can also be used for collecting and transporting the secreted factor
9 1.3 Evidence of a Role for Brassinosteroid Signaling in C. roseus Carpel Fusion. While ma ny of the signaling events that initiate and coordinate the fusion process remain a mystery, rece nt work has demonstrated a likely role for brassinosteroid hormones (Kahn, 2006; Goodman, 2012; Clore et al, in revision). Buds treated with Brz2001, a spec ific inhibitor of brassinosteroid biosynthesis (Sekimata, 2001) exhibit abnormal carpel fusion wherein the carpels a d here superficially, but deeper, more s ubstantial fusion is absent. T he two carpels are easily separated when probed with a dissecting need le (Kahn, 2006; Goodman, 2012; Clore et al, in revision) The effect can be rescued by application of an exogenous brassinosteroid (Goodman, 2012, and Clore et al, in revision). Furthermore, Goodman (2012) showed that carpel exudates captured in agar elici ted a brassinosteroid like response in a wheat leaf unrolling bio assay (Wada, 1984). First isolated from Brassica pollen, brassinolide is a representative member of the brassinosteroid family, with the characteristic four ring structure found in animal st er oids (Groves, 1979). Its small size is con sistent with the properties of the diffusible fusion factor predicted by earlier studies of C. roseus (Verbeke and Walker, 1986). The somewhat hydrophobic nature of the molecule may make it a good candidate for diffusing across the thin layers of cuticle and pectin which appear to persist across the fusion plane (Walker 1975b.) Other common and d iffusible plant hormones have been tested for factor like activity, namely cytokinin (Goodman, 2012), auxin, and gibber ellic acid (J. Verbeke, personal communication to A. Clore) but these were not found to contribute to the fusion response.
10 1.4 Overview of the Brassinosteroid Signaling Pathway in Plants Brassinosteroid action in other plant systems has been establishe d as an important regulator of stem elongation, cell division and differentiation, anther and pollen development, and various stress responses (Mandava, 1988; Kim, 2010; Clouse, 2011). Unlike animal steroids, which often diffuse across the cell membrane to interact with cytoplasmic and nuclear receptors, brassinosteroid signaling in plants begins with binding of the steroid to the membrane bound receptor BRI1 (BRASSINOSTERO ID INSENSITIVE 1, Clouse, 1996). The receptor is a member of the leucine rich repeat receptor like kinase family, with structural and functional similarities to animal receptor tyrosine kinases (Li and Chory, 1997). Ligand binding relieves the constitutive inhibition of the brassinosteroid response pathway, unleas h ing a complex series of i nteractions mediated by cytosolic kinases and phosphatases. The signal ultimately reaches the nucleus and ef fects changes in gene expression (Clouse, 2011; Yang, 2011). While the vast majority of the studies illuminating the signaling events have been cond ucted on Arabidopsis thaliana proteins homologous to major players in the signaling pathway have been found in numerous other sp e cies including rice (Shiu, 2004), tobacco (Nakashita, 2003), maize (Hartwig, 2011), and pea (Nomura, 2007). These and other ex amples indicate that the pathway is likely kingdom wide. A brief overview of the steps in this pathway follows ( see also Figure 1.5) Brassinosteroid ligands first bind to a hydrophobic groove in the extracellular domain of BRI1, activating an induced fit mechanism and altering the structure of the protein immediately around the binding site (She, 2011). This structural rearrangement
11 Fig. 1.5: The brassinosteroid signal transduction pathway, with (bottom) and without (top) bras sinolide ligand binding (Kim, 2010). Unlike animal steroids, plant steroid action depends on the molecule binding to an extracellular receptor, whereupon a series of phosphorylation and dephosphorylation reactions relieve the constitutive inhibition of sig nal transduction and allow for the transcription of key brassinosteroid response genes. The pathway is described in further detail in the text.
12 enables the phosphorylation of BKI1 (BRI1 KINASE INHIBITOR 1) by BRI1, which interrupts a plasma m embrane targeting motif on BKI1 and leads to its dissociation from the membrane (Jallais, 2011). Whereas membrane localized BKI1 binds to BRI1 and inhi bits its signaling capabilities, phosphorylation mediated dissociation from the membrane relieves this in hibition (Jallais, 2011). Subsequently, BRI1 is free to associate with its with its coreceptor BAK1 (BRI1 ASSOCIATED KINASE 1) (Russinova, 2004; Kim, 2010). BRI1 and BAK1 transphosphorylate each other on tyrosine residues, leading to complete activation of the signaling pathway (Wang, 2008). A family of homologous membrane associated proteins known as BSKs (BRASSINOSTEROID SIGNALING KINASES) have been identified as substrates of BRI1 (Tang, 2008). These kinases dissociate from the BRI1/BAK1 receptor complex upon phos phorylation and act as positive regulators of the brassinosteroid response pathway by phosphorylating downstream cytoplasmic effectors (Tang, 2008; Clouse, 2011). BSK1 has been shown to phosphorylate and activate BSU1 (BRI SUPRESSOR 1), a phosph atase which inactivates BIN2 (BRASSINOSTEROID INSENSITIVE 2) (Kim, 2010; Clouse, 2011). BIN2 is a member of the GSK3/SHAGGY like kinase family and has long been recognized as a negative regulator of brassinosteroid responses (Li and Nam, 2002). Activ e BIN2 phosphorylates the brassino steroid response transcription factors BZR1 and BES1, preventing their nuclear localization (Ryu, 2010). BZR1 and BES1 are bound to cytoplasmic 14 3 3 proteins in their phosphorylated state ( Gampala, 2008), which reduces DNA bin ding capability (Kim, 2010). Phosphorylation thus inhibits the nuclear action of the transcription factors by altering their localization and structure, as well as targeting them for proteasome mediated degradation (Kim, 2008). Inactivating
13 BIN2 relieves t he inhibition of transcription and allows the activation of brassinosteroid response genes, more than 500 of which have been identified (Yang 2011). Response specificity is achieved through supplemental transcription factors, often bHLH proteins (Clouse 2 011), and through BIN2 mediated interplay between the brassinost eroid signaling pathway and those of other hormones, including abscissic acid and auxin (Saidi, 2012). 1.5 Brassinosteroids and Plant Development The canonical role s for brassi nosteroids a re in promoting cell division, expansion, and tissue elongation (Mandava, 1998; Kim, 2010; Clouse, 2011). However, several studies have demonstrated that brassinosteroid signaling can also be an important regulator of cellular differentiation as well as o f epidermal and floral development. One example is the morphogenesis of tracheary element s. This process begins with d edifferentiation from mesophyll tissue to less specialized cells, followed by a reduction in potency and a change to tracheary precursor c ells and, finally, the formation of the secondary cell wall (Fukuda, 1997). In Zinnia elegans, these developmental changes are preceded by a marked increase in intracellular levels of several bioactive brassinosteroids, and application of a brassinosteroid biosynthesis inhibitor prevents normal tracheal development (Yamamoto, 2001). Brassinosteroids thus have a previously described role in cellular redifferentiation. Brassinosteroid hormones also appear to play a part in cell to cell signaling in epiderm al tissue Adjacent cel l ce l l communication determines the pattern of haired and hairless epidermal cells in Arabidopsis roots, and brassinosteroids have been shown to be
14 an important mediator of cell fate in this system (Kuppusamy, 2008). Proper epidermal patterning depends on the lateral movement and position dependent accumulation of transcription factors that inhibit the hair cell pathway and indirectly promote the development of non hair cells (Larkin, 2003). Brassinoster oid treatment increases the exp ression level of key transcription factors, and bri1 mutants as well as roots treated with inhibitors of brassi nosteroid biosynthesis exhibit abnormal patterning of haired and hairless cells (Kuppusamy, 2008). Brassinosteroid signals per ceived in the epid ermis can coordinate responses in other tissues. Brassinosteroid insensitive or defic ient dwarf mutants were transformed with brassinosteroid receptor or biosynthesis genes targeted exclusively to the epidermis through the use of a site specific promoter. I n both cases the plant was rescu ed from the dwarf phenotype (Sa va l di Goldstein, 2007). These findings suggest that brassinosteroids play a role in relaying non autonomous signals, wherein signals perceived in the outer epidermal layer are transferred to t he ground tissues responsible for growth. Interestingly, it was shown that cell to cell movement of the transcription factors BES1 and BRZ1 was only partially responsible for generating a growth response in sub epidermal tissue, suggesting that additional downstream transcription factors that are not part of the general brassinosteroid response pathway are also involved (Saval di Goldstein, 2007.) Also pertinent to brassinosteroid signaling is the growing body of evidence that related molecules play a role in floral patterning and morphogenesis, at least in Arabidopsis. Proteins in the GSK3/SHAGGY like kinase family, with a high level of structural homology to BIN2 (which negatively regulates the brassinosteroid response pathway in the absence of BR signalin g), are also necessary for proper floral
15 development (Dornelas, 2000). Antisense mutations of two related proteins, AtSK11 and AtSK12 ( ARABIDOPSIS SHAGGY RELATED KINASE 11 and 12), were examined in Arabidopsis plants These mutants display a phenotype with several disruptions in floral structure, including unfused carpels and altered epidermal cell fate on the abaxial side of these carpels (Dornelas, 2000). Furthermore, more recent work on the brassinosteroid signaling cascade used a yeast two hybrid assay to demonstrate that AtSK11 and AtSK12 can interact with with BRZ1, presumably in a similar manner to BIN2 (Kim, 2009). The interaction was verified in vivo with a bimolecular fluorescence complementation ( BiFC ) assay, wherein one half of yellow fluorescent protein molecules were fused to BRZ1 and the other half to AtSK12. When both constructs were expressed in Arabidopsis seedlings the yellow fluorescent protein fluoresced normally, indicating that the two molecules were in close enough proximity for the pi e ces of YFP to reunite and assume a normal conformation. The results help explain the persistence of phosphorylated BES1 and BRZ1 even when the genes coding for BIN2 and closely related kinases are knocked out (Vert, 200 6; Kim, 2009). These studies indicate that proteins involved in the brassinosteroid signaling cascade are indispensable for the normal development of reproductive structures and, more broadly, that a wide range of redundant kinases, many of them also enmes hed in other pathways, help to relay brassinosteroid signals from the cell surface to the nucleus. Sterols are a class of molecules which form the biosynthetic precursors to brassinosteroids. They are also important structural components of cell membranes influencing permeability and stiffness and helping to modulate the function of membrane bound proteins (Schaller, 2004). The identity and biological function of sterols is
16 determined by a class of enzymes known as sterol methyltransferases (SMTs). Arabid opsis SMT mutants have unfused carpels and abnormal planes of cell division, among other morphological defects, suggesting that these molecules play a key role in plant development (Carland, 2010). Biochemically, the mutants do not display a reduction in t he overall level of brassinosteroid precursor, but rather alterations in the levels of several unique sterols relative to one an other. Additionally, the enzymes rendered ineffective in the mu tants act at a branch point between sterols and bras sinosteroid s ynthesis, so brassi nosteroid production should proceed normally even in the mutant plants. External application of synthetic brassinosteroid failed to rescue the effects of the SMT mutants. This collectively points to phenotypic effects that are brassinost eroid independent, and suggests that the ratio between key sterols is important for proper development (Carland, 2010). Sterols have also been linked to plant cell polarity through their role in mediating polar auxin transport: SMT mutants show defects in cell polarity, abnormal auxin distribution, and mislocated PIN proteins (Willemsen, 2003). While no evidence has yet been reported linking altered sterol composition to carpel fusion in C. roseus the apparent role of sterols in Arabidopsis gynoecial devel opment and their capacity for altering plasma membrane properties make them an intriguing possibility. Taken togethe r, the above examples provide precedence for brassinosteroid action (and that of other sterol s ) in determining cell fate and in driving re differentiation. They show that brassinosteroids can act specifically on epidermal cells, and that related molecules appear to be essential for development of normal carpel morphology in Arabidopsis P ostgenital carpel fusion in C. roseus is mechanisticall y unique and much
17 more complex than the congenital fusion that occurs in Arabidopsis as disc ussed above. However, Arabidopsis studies have contributed greatly to our understanding of the brassinosteroid signal transduction pathway and appreciating the s cope of brassinosteroid systems. While there is convincing evidence that brassino steroid signaling is an indisp ensable part of C. roseus carpel fuision the intrica cies of just how cellular and morphological changes are effected and what other signaling pathways may play a part have yet to be uncovered. 1.6 A Case C. roseus Carpel Fusion In addition to sugges ting a role for brassinoster oid signaling in regulating the postgenital carpel fusion of C. roseus recent work has characterized changes in cuticle permeability that take place over the course of the fusion process (Goodman, 2012; Clore et al. in revision). The cuticle (F igure 1.6) is a thin layer covering the above ground surfaces of plants, which functions in protection and in preventing excessive water loss. It is composed of the polymer cutin and various cuticular waxes, and is hydrophobic and highly lipophilic (Samuels, 2008). Cuticular permeability assays using TBO (toluidine blue O) and ruthenium red revealed distinct, reproducible staining patterns corresponding to different stages of fusion. TBO and ruthenium red stains pectins (Curvers, 2010 ). Since these are both components of the cell wall, persistent staining indicates that the dyes have crossed the barrier of cuticular wax and thus correlates with cuticle permeability, as has been verified by several researchers (including Tanaka, 2004; B essire, 2007; and Curvers, 2010).
18 Permeability assays using these two dyes conducted by Goodman (2012) showed that prefusion C. roseus carpels with a visible space between them were resistant to staining, indicating impermeable cuticle at this stage. The carpels begin to stain lightly around their base after making contact but prior to initiating fusion, particularly on the adaxial (inner) surfaces. Carpels which are tightly appressed and actively fusing stain around the gynoecium on both the inner and ou ter surfaces, but remain unstained at the distal tips. As the style elongates in the postfusion gynoecium, heavy staining is observed in the developing skirt and the stigmatic region, but is much fainter at the distal tips (Goodman, 2012; Clore et al., in revision). Staining patterns indicative of increased Fig. 1.6: A generalized illustration of the plant cell cuticle. Epicuticular waxes (A) protrude from the surfac e of the epidermis. The cuticle proper (B) contains the polymer cutin, as well as intracuticular waxes. The cuticular layer (C) also contains cutins and waxes, and is contiguous with the pectinaceous middle lamella. The cell wall and the cytoplasm are repr esentd by (D) and (E), respectively. Cuticle permeability depends on the identity and arrangement of intracuticular waxes, as discussed in the text. (Adapted from Buchholz, 2006).
19 permeability are therefore correlated with regions of the carpel that are competent to fuse. Adaxial surfaces in the stained region undergo fusion during normal development, and the circumferential are as which also stain can be made to adopt a fusion fate through the exogenous application of signaling factors, as described earlier in this discussion. Carpels which have not yet begin to fuse, and areas such as the very distal rounded tips and which remai n un fused throughout development do not stain (Goodman, 2012; Clore et al., in revision). The permeability of the cuticle depends on the properties of the molecule (s) attempt ing to cross and on the thick ness and composition of the cuticle itself, which v aries widely across species and developmental stages (Schreiber, 20 05; Nawrath, 2006; Yeats, 2012) The density and composition of waxes packed into the cutin scaffolding appear to determine the transport properties of the cuticle, especially when the pene trating molecules are non polar (Buccholz, 2006; Bessire, 2007). Many mutant phenotypes with an altered cuticle also display significant d ifferences in a variety of functions These include water stress response and the abscis s ic acid pathway (Wang, 2011) reproduction and gamet ogenesis (Smirnova, 2013) and plant defense and re sponse to infection (Bourdenx, 2011) Cuticle mutants often exhibit abnormalities in organ separation which will be discussed below. These lines of evidence suggests a variety of i mportant and likely interrelated roles beyond the mere regulation of water loss, for the cuticle itself and for enzymes active in its biosynthe sis. Of particular note are s everal mutant phenotypes with a compromised cuticle which show increased resistance normally protective role, it is somewhat surprising that enhanced cuticle permeability
20 would correlate with resistance to pathogen attack. However, such a response has been reported in Arabidopsis mut ants interacting with Botrytis cinerea a necrotro phic fungus ( Bessire, 2007; Curvers, 20 10), and Pseudomonas syringae, a bacterium which is often responsible for frost damage in plants (Xiao, 2004). It has been well established that plants rely on molecu lar signals (known as pathogen associated molecular patterns, or PAMPs) presented by invaders to trigger their innate immune response and kick start pathways that lead to resistance genes and other plant defense mechanisms (reviewed in He, 2007). These sig nals are usually small molecules unique to either bacteria or fungi and may be proteinaceous, (i.e. flagellin; EF Tu) or components of the pathogen cell wall or outer membrane (such as chitins, glucans, or lipopolysaccharides; as reviewed in Nicaise, 2009) PAMPs bind to membrane spanning pattern recognition receptors, thereby activating intracellular pathways leading to increased immunity (He, 2007; Nicaise, 2009). Given the importance of small molecule signaling at the cell surface to the plant immune re sponse, it has been hypothesized that heightened pathogen resistance in cuticle biosynthesis mutants is due to a more efficient percep tion of pathogenic cues (Bessire, 2007) The cases highlighted above represent intriguing examples of how altered cuti cle permea bility interacts with signaling. The mutations in qu estion interfere with cutin bio synthesis and wax deposition via nonfunctional versions of enzymes responsible for the synthesis and oxidation of long chain fatty acids, which are biochemical pre cursors to cutin and other surface waxes (Xiao, 2004; Bessire, 2007; Curvers, 2010). Given that the enzymatic pathways involved in cuticle synthesis and modulation are complex and still being deciphered (i.e. Samuels, 2008), the ability of these pathways to
21 have far reaching effe cts on other signaling events enables another layer of potential cross talk. In the case of carpel fusion in C. roseus the effectors evidently exchanged between developing carpels are similar to PAMP molecules in t hat they too, m ust presumably be perceived by r eceptors at or near the plasma membrane Since the carpel surface cells that eventually fuse are initially epidermal in nature and evidently covered with a cuticle similar to that found on other epidermal surfaces (Walker 19 75b), it is a reasonable hypothesis that similar alterat ions in cuticle perme ability occur in this instance over the course of normal development This hyp othesis was originally proposed by Lolle (1999), based on studies with Arabidopsis mutants displaying organ fusions similar to that of C. roseus carpels, and it has been supported by the recent work in the using TBO and rhuthenium red assays, as described above (Goodman 2012; Clore et al, in revision) Considering the interactions between cuticle permeabi lity and signaling events in othe r systems may help illuminate the role of these permeability changes in C. roseus development. Several genetic and biochemical studies on Arabidopsis mutants displaying abnormalities in either organ separation and/ or epider mal patterning suggest an important cuticular role in these processes. A molecular gene tics approach is often employed. In this type of study, the sequence abnormalities of mutations with an observed phenotypic effect are isolated and characterized. This process links genetic sequence, protein product and the mutant phenotype that results from the alteration of a single gene. FIDDLEHEAD was one of the first cuticular genes to be thus investigated (Lolle, 1992). Fdh mutants lacking a functional version of the FIDDLEHEAD gene, exhibit superficial, surface level fusion be tween their flora l organs. For example, sepals and petals may fuse
22 either within a single bud or between adjacent buds to create a messy conglomeration and trap stamen s within the fused tissu e (Lolle, 1992) This fusion is similar mechanistically to the very first superficial stage of the fusio n found in C. roseus carpel development but lacks the subsequent cellular redifferentiation The fusion suture persists in the fiddlehead plants, in c ontrast to normally developing C. roseus carpels where the previously epidermal cells become indistinguishable from the surrounding tissue (Lolle, 1997 ; Walker, 1975a ). Fdh plants are also noteworthy for their ectopic pollen response Pollen grains that come into contact with l eaves of fdh mutants will become spherical and fully hydrated within minutes, mimicking the normal response of pollen grains landing on the stigma In non mutant plants this pollen hydration reaction is restrict ed to papillary sti gmatic cells and is highly specif ic (Zinkl, 1999) Following hydration, pollen forms a cytoplasmic outgrowth known as the pollen tube which extends down the stigma and into the ovaries, enabling fertilization (Swanson, 2004). The Arabidopsis stigma will hydrate only Arabidopsis pollen, and the reaction depends both on the complementary (interlocking) structure of the pollen and stigma surfaces and also on lipophilic adherence molecules that mediate the interaction (Lolle and Pruitt, 1999 ; Zinkl, 1999 ). T he adherence molecules are carried within the pollen wall and prompt both attachment and hydr ation from the stigma, which is a dry surface and remains dry until it encounters compatible pollen (Edlund, 2004) P ollen response is therefore a significant exam ple of an otherwise inert surfaces experiencing a contact mediated recognition and subsequently exchanging signals and becoming responsive to one another. The fdh mutation extends this responsiveness to leaf epidermal surfaces.
23 Both the abnormal fusion o f floral organs and the ectopic response to pollen grains characteristic of the fiddlehead mutation a re reminiscent in some senses of the carpel fusion that occurs as a part of normal gynoecial development in C. roseus In both cases previously inert epid ermal surfaces become competent to interact with one another, and eventually adhere. Additionally, fdh mutants show altered cuticle properties and an increased permeability to both chlorophyll and chemical fixatives ( Lolle, 1997), similar to the permeabili ty changes observed previously in the carpels (Goodman 201 2 ) These similarities suggest that the mutagenesis approach to elucidate the genes and gene products involved in the fdh mutation may have some relevance to the question of C. roseus carpel fusion The FIDDLEHEAD gene has been found to encode a ketoacyl CoA synthase, which catalyzes the synthesis of very long chain fatty acids (Pruitt, 20 0 0). A related gene HOTHEA D, has also been described (Krolikowski, 2003). T he mutant phenotype manifests similar effects such as abnormal organ fusion, incre ased cuticle permeability and ectopic pollen hydration (Krol ikowski, 2003). HOTHEAD is hypothesized to code for a metabolic oxidoreductase enzyme that is ultimately instrumental in the synthesis of long chain fatty acids (Kurdyokov, 2006). The organ fusi on mutants described above suggest the possibility of an important role for the cuticle in regulating C. roseus carpel fusion The effect of cuticle permeability on the efficien cy of small molecule signaling likewise supports this hypothesis, as do permeab ility assays conducted over the course of the fusion process. A diverse set of enzymes have been shown to be instrumental in the cuticle biosynthesis pathway and many of these produ ce an altered cuticle phenotype when mutated. I t seems likely that the rel ative levels of myriad lipophilic products have an effect on the properties of the
24 cuticle. Cuticular properties are regulated by a complex set of intersecting pathways, which evidently interact with oth er signaling events in a signifi cant manner. These pa thways present an intriguing avenue of investigation for deciphering carpel fusion in C. roseus one which to date has been little explored. 1.7 Hypothesized Role s for Reactive Oxygen Species Signaling in Carpel Fusion in C. roseus At least one study h as linked a permeable cuticle to the release of reactive oxygen that are important in plant biology include hydrogen peroxide ( H 2 O 2 ), superoxide (O 2 ), and hydroxyl radical (OH ), among others (Bhatt acharrjee, 2012). They have a variety of cellular sources, including electron transport chain s in the mitochondria and chloroplasts, and a family of membrane bound NADPH dependent oxidase enzymes (Finkel, 2011). A com monality among ROS is their abil ity to cause rapid and severe oxidative damage to proteins, lipids, and DNA (Apel, 2004); for this reason t hey were long regarded as predominantly damaging cellular by products, with some useful roles in plant defense (Doke, 1996; Bolwell, 1997) However, a grow ing body of evidence has led to the acceptance of a complex and indispensa ble role for R OS in signaling networks (Mittler, 2011; Van Norman, 2011 ; Bhattacharjee, 2012). Examples of ROS signals driving morphological changes in epidermal cells are particular ly interesting when considering additional candidate pathways for the regulation of C. roseus carpel fusion especially given increasing reports of redox status contributing to cell fate determination and the establishment of tissue polarity (reviewed in V an Norman, 2011 and Clore, 2013 ).
25 One mutant defici ent in cuticular wax production and exhibiting heightened cuticle p ermeability was also found to have increased ROS prod W ounding (perforation of the cuticle and epidermis) and t he presence of pathogens are well known to trigger an oxidative burst that likely functions both in signaling and in defense against biotic stressors (Ap el, 2004). (2011) reported elevated ROS levels in two Arabidopsis cuticle mutants, bo dyguard (bdg) and lacs1 which ar e deficient in cutin organization and wax biosynthesis, respectively. ROS levels were measured by quantifying dichlorfluorescein diacetate ( DCF DA ) fluorescence an established H 2 O 2 detector. Thet were found to be constitu tively higher than wild type plants in both lacs and bdg mutants. In contrast to wild type plants, f luorescence in the cuticle mutants increased further when the wound site was treated with either water or a mock solution of dextrose broth, as well as when the wound was inoculated with B. cinerea spore suspension (L'Haridon, 2011). The higher levels of ROS in cuticle mutants even in the absence of pathogenic cues suggests that cuticle structure and the redox status of the cells may be connected in ways th at have not yet been fully explored. Since the cuticle covering C. roseus carpels undergoes alterations in its per meability throughout fusion it makes sense to consider whether associated changes in ROS levels might play a signaling role in this process C. roseus carpel fusion also has potentially meaningful similarities to pollen stigma interactions, as described above, and this is another process where ROS signaling has been previously implicated. Peroxidase enzymes that are specific to the stigma hav e been isolated and characterized (McInnis, 2006), and mature (receptive) stigmatic papillary cells con s titutively produce high levels of H 2 O 2 and other ROS. This response appears to lessen once pollen adheres suggesting
26 an intriguing potential for ROS a and stigma cells to interact with one another (McKinnis, 2006; Hiscock, 2007). Furthermore, the antioxidant glutathione, which is an important mediator of redox state in plant cells (Foyer, 2011), is a necessary factor for pollen germination (Zechmann, 2011). B rassinosteroid signals exchanged between developing carpels have been demonstrated through several lines of evidence to be necessary for normal carpel fusion in C. roseus. In stress response al so mediated by brassinosteroid signaling, the hormones induce ROS accumulation through the up regulation of NADPH oxidase genes (Xia, 2009). The application of ROS scavengers disrupts the stress resistance that is otherwise induced by BR signaling. The ex act mechanism through which this mediation occurs remains unknown, but it is hypothesized that perception of the BR signal activates NAPDH oxidases, either through transcriptional control or via an extra nuclear route possibly involving the BRI1 co recept or BAK1. The resulting increase in ROS may activate a protein phosphorylation cascade, ex tending the signaling repertoire and eventually activating several stress response genes (Xia, 2009). Whatever mechanisms are in fact at play, the stress response prov ides precedence for interaction between the BR and ROS signaling pathways. 1.8 ROS Signaling In Plant Development, and P otential Mechanisms of Action The examples just discussed present key possibilities where ROS signaling might intersect with signal ing events that have demonstrated importance in C. roseus carpel fusion. More generally, a wide variety of evidence implicate s ROS signaling and/or cellular redox status as important carriers of information during various processes of
27 plant development. Th e formation of adventitious roots for example, involves the redifferent iation of mature vasculature into meristematic tissue. T he initiation of this process is mediated by H 2 O 2 in a concentration dependent manner (Li, 2009; Bai, 2012). Hydrogen peroxide signaling in this case appears to interact with auxin and nitric oxide to induce an accumulat ion of cyclic GMP and eventually a change in morphogenetic fate, with determined cells giving rise to undifferentiated root primordia (Bai, 2012). ROS distribut ion is often visualized using diaminobenzidine (DAB).This indicator is oxidized by hydrogen peroxide in the presence of endogenous NADPH oxidase to form a brown precipitate, and can thus be used to detect one of the most common reactive oxygen species. The technique has been used by several researchers, including Thordal Christensen (1997), and Ramel (2009) Kwasniewski (2013) used this staining method, among other techniques, to show that ROS production is correlated with root hair initiation and elongatio n in barley (Kwasniewski, 2013). Peroxidases and oxidases specific to root hair cells are thought to work together in a coordinated manner, first loosening cell walls for a localized initiation of root hairs and then aiding in the establishment of a Ca + gr adient that directs their elongation (Monshausen, 2007; Kwasniewski, 2013). Both of these instances of organogenesis lend credibility to the idea that ROS can both drive differentiation and aid in the establishment of tissue polarity, a fundamental featur e of plant biology that is necessary for any sort of asymmetric growth or specialization (Grebe, 2004). Another situation where the establishment of polarity is par amount is in gravitropism, the directional growth response to gravity. This phenomenon has been studied extensively both in roots (Boonsirichai, 2002), and in pulvini, specialized stem
28 organs which contribute to upright growth in certain stems (Clore, 2013). ROS signaling has been found to play a role in both these processes, where it mediates t he asymmetric elongation necessary for curved growth. In roots, ROS accumulate in the lower cortex of the tip in response to gravistimulation, and exogenous application of hydrogen peroxide induces root curvature towards the application (Joo, 2001 ). The di fferential accumulation continues for up to three hours after the root tip is turned (changing its orientation necessitates a change in the direction of growth), and the application of ROS scavengers inhibits to gravitropism response. In maize stem pulvini similar gravistimulation experiments as well as the visualization of ROS distribution within the tissue led to the conclusion that the establishment of an ROS gradient, the orientation of which is determined by the gravity vector, may provide directional cues for subsequent growth and elongation (Clore, 2008). It is clear that ROS represent a diverse set of mechanism by which signals can be transduced between and within cell s, and which can convey directional and developmental information that leads to c hanges in cellular response. The above anges in ROS levels and the redo x status of cells or tissue lead to a differential response is currently an area of active research, and several intriguing possibilities present themselves. In the maize gravitropism response, ROS accumulation was accompanied by increased transcription of an aconitas e /iron regulatory protein 1 homologue (Clore, 2008 ). Aconitase is an iron sulfide cluster pr otein which has a high sensitivity to ROS due to the oxidative inhibition capacity of these molecules (Navarre, 2000). Proteins inhibited by ROS, or whose action can be modulated by oxidation, could play a role in
29 interpreting ROS gradients in the cell and in translating these fluctuations into far reaching effects Sensitivity to redox state has been demonstrated on the part of many proteins with thi ol containing domains especially those with two or more cysteines O xidation of these residues can generat e a disulfide bond and form a stable product with altered biochemical activity (Foyer, 2005). Thioredoxins and glutathiones are a particularly important example of this in both plant and animal sys tems (Fujino, 2006 ; Finkel, 2011 ). R eports of a membrane a ssociated thioredoxin capable of intercellular movement in plants (Meng, 2010 ) suggest that these and related molecules could have an important role in cell to cell communication. Transcri ption factors that can both respond to and influence changes in leve ls of ROS have also been proposed. In Arabidopsis (and many other plant species), the root apex consists of both the rapidly dividing meristem and the elongating cells just behind it; these two regions are demarcated by the transition zone (Baluska, 2010) The transition zone represents a boundary between proliferation (in the meristem) and differentiation (in the zone of elongation). Research suggests that the trans ition zone also marks a border between two distinct ROS environments, with accumulation of O 2 coinciding with proliferation while differentiation appears to require increased levels of H 2 O 2 (Tsukagoshi, 2010). ROS balance is mediated by peroxidase enzymes which have the dual function of reducing hydrogen peroxide and catalyzing the productio n of superoxide. The expression of peroxidases along the transition zone is regulated by a mobile transcription factor, which is in turn can be up or down regulated in response to levels of H 2 O 2. The identification and characterization of this transcripti on factor suggests a
30 complex system of feedback loops which regulate redox homeostasis and, by extension, the balance between cellular proliferation and differentiation, a regulation which is essential for proper growth and organogenesis (Tsukagoshi, 2010) Changes in ROS levels can be linked to protein phosphorylation, and thence to a dizzying array of signaling networks, through the oxidation of methionine residues (Hardin, 2009). Oxidation of active site methionine residues in both calcium and cAMP dep endent protein kinases resulted in a marked inhibition of the phosphorylation function, at least in vitro In vivo the exogenous application of hydrogen peroxide influenced the phosphorylation of nitrate reductase in a conc entration dependent manner. L ow concentrations of H 2 O 2 increased phosphorylation rate and higher concentrations inhibited the process (Hardin, 2009). The model of methionine oxidation as a signaling mechanism, and not solely a by product of oxidative damage, is further supported by the fact that cyclic GMP can also induce methionine oxidation at levels comparable to hydrogen peroxide (Marondedze, 2013). These findings suggest the existence of cellular machinery to carry out methionine oxidation in response to small molecule signaling. c GMP is a widely used signal active in myriad cellular processes, and any process that utilizers this molecule indicating likely has a specific role in larger signaling network s (Marondedze, 2013). T he potential oxidation of methionine residues on the BRI 1 receptor kinase is being investigated as a possible link between ROS and brassinosteroid signaling (Det er, 2011). These diverse examples of ROS signaling illustrate its importance in plant development and directional response They likewise provide a co ntext in which to examine how this signaling network might contribute to the establishment of tissue
31 polarity or the transduction of contact mediated signals during the carpel fusion process. New roles for the ROS signaling network, as well as the mechanis m s which function in the perception of and response to these signals, are the subject of ongoin g research. Exploring ways in which ROS signaling could interact with established and/ or as yet unknown regulator s and mediators of postgenital carpel fusion may provide insight into this unique process, and potentially into broader themes of intercellular communication, coordinated development, and redox status as an information carrier in plant cells. Section 1.9 Thesis Overview This introductory revie w of l iterature has attempted to first provide a foundation of what is currently understood about carpel fusion in Catharantus roseus Morphological changes, including the contact mediated nature of the fusion and the loss of epidermal character of cells along t he fusion sutu re, have been well documented by past researchers As reviewed earlier, p revious work has established that an exchange of diffusible fusion factors between developing carpels is necessary for the fusion process, and that brassinosteroid hormo nes represent at least one component of this chemical exchange. Permeability assays have shown that reproducible changes in cuticle permeability occur throughout the course of fusion and reactive oxygen species signaling, while not yet explored in C. rose us fusion, is important in many aspects of plant development in particular those necessitating directional cues. The current study attempted to elucidate the possible link between brassinosteroids and the surface structure of the fusing carpels. Carpels were allowed to develop on C. roseus cuttings grown in the presence Brz2001, a specific brassinosteroid
32 bio synthesis inhibitor These brassinosteroid deficient carpels were examined using scanning electron microscopy (SEM) throughout development, providing a detailed look at the patterns of cells on prefusion, fusing, and postfusion surfaces. The Brz2001 treated carpel surfaces were compared to those from untreated and solvent control treated cuttings. In a similar experiment, cuttings were treated with an inhibitor of NA D P H oxidase, one of the major cellular sources of hydrogen peroxide. Carpels from these buds were examined with light microscopy and SEM to ascertain whether this inhibition of one important ROS source interfered with fusion, and the effects of the inhibition on surface structure over the course of development in particular. Finally, buds at various stages of fusion were treated with DAB stain in an attempt to visualize and localize patterns of ROS production and/or accumulation.
33 Chapter 2: Materials and Methods 2.1 Cultivation Catharantus roseus flats from a local nursery. The plants were kept in a growth chamber under fluorescent light s on a 16 h day/8 h night cycl e and maintained at 25 C. Seedlings were transferred The soil mixture consisted of peat moss, vermiculite, potting soil, sand, and pearlite in a 4:4:1:3:2 ratio. Plant s were watered three times per week, and with each watering received 150 ml Southern Ag Powerpak 20 20 20 fertilizer solution. 2 .2 Treatment with Brz2001 and D iphenyleneiodonium Treatment with the brassinosteroid biosynthesis inhibitor Brz2001 was previously shown to impair carpel fusion (Goodman, 2012; Clore et al. in revision). To examine the effects of this inhibitor on the fine surface structure of developing carpels, live buds were treated with Brz2001 as described previously (Goodman, 2012) and below Buds were also treated with diphenyleneiodonium (DPI) to explore the role of ROS signaling in C. roseus carpel fusion, and the effects of this signaling pathway on carpel surface structure in particular. DPI inhibits NADPH oxidase, a major sourc e of ROS in plant cells (Vianello, 1991; Qin, 2004). Cuttings were taken from healthy plants grown as described above, and selected for apical regions containing several pre fusion buds. Scintillation vials were prepared with 22 ml of a 10 M solution of ei ther Brz2001
34 (courtesy of T. Asami, University of Tokyo, Japan) or DPI (Sigma Aldrich, St. Louis, MO USA) dissolved in dimethyl sulfoxide (DMSO, Sigma Aldrich, St. Louis, MO, USA) and diluted with de ionized water. Concentrations of DPI and Brz2001 were selected to insure adequate inhibition without demonstrating toxic effects as reported in Jiang (2002) and Clore (20 08) for DPI, and in Goodman (2012) for Brz2001 Jiang (2002) additionally gave precedence for DPI uptake into cells via cut stems in soluti on, and showed that the inhibitor remains active for a period of several days at typical growth chamber temperatures. Solvent control vials with 22 ml of 0.1 % (v/v) DMSO were also prepared. The vials were capped with Parafilm into which a hole was poked to allow the insertion of cuttings, and wrapped in foil to protect the chemicals from light degradation. Stems were cut at an angle 4 5 cm down from the apical buds re trimmed underwater to prevent embolism formation, and inserted immediately into the par afilm capped vial. Buds that were prefusion at the start of the treatments were marked with ink on the tips of the sepals to facilitate later identification of those which had developed in the presence of inhibito r. Buds were allowed to develop on the cu ttings in solution which were incubated in the growth chamber under identical conditions to those described for whole plants above. After two weeks, marked buds were h arvested and fixed in 2% gluta raldehyde made from stock 8 % glutaraldehyde ( Ted Pella, I nc., Redding, CA, USA ) diluted in phosphate buffered saline (PBS: 0.1 M phosphate buffer with 0.0025 M potass ium chloride and 0.137 M sodium chloride at pH 7.4) Fixation continued for a minimum of 4 h on a tissue rotator. The buds were rinsed three times with phosphate buffered saline and then dehydrated in a series of ethanol washes of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 1 00% (v/v) ethanol for 15 min each, also on a
35 rotator Fixed buds were stored in 10 0% ethanol at 4C until they were dried and coated for visualization with the scanning electron microscope (SEM). 2.3 Tissue Preparation and Drying Carpels were examined with a Topcon SEM in order to characterize any changes that may occur to their fine surface structure during development. Samples must be thoroughly dry to give good SEM images and to avoid contamination of the vacuum column Since conventional drying risks damage to delicate tissues due to high surface tension at the air water interface as water evaporates, critical point d rying was employed. This method makes use of the f act that at a temperature of 32 C and a pressure of 1200 gaseous state with no change in surface tension (Electron Microscopy Sciences, 2013) Liquid can thus be evacuated from the sample without the risk of tissue damage. Prior to the drying procedure, f ixed and dehydrated buds stored in ethanol were bathed in a series of 25%, 50%, 75%, and 100% (v/v in ethanol) amy l acetate (Electron Microscopy Scienc es, Hatfield, PA, USA) for 15 min each. Amyl acetate is miscible with both water and CO 2 and so is a suitable intermediate liquid. A Polaron E300 Critical Point Drying Aparatus (Quorum Technologies, Ltd, East Grinstead, West Sussex, United Kingdom) was used to complete the drying process. The buds were kept under 100% amyl acetate and placed in the specimen holder in the drying chamber of the critical point dryer. The chamber was filled with liquid CO 2 and the drain valv e opened slightly to maintain a constant level of liquid, flushing out the amyl acetate and replacing it with CO 2 This flushing act ion was kept up for 5 min after which time the drain valve was
36 closed and the chamber allowed to fill with liquid CO 2 Sam ples were incubated in CO 2 for 1 h followed by a 5 min flush and a second 60 min CO 2 incub ation. After a final 5 min flush, the temperature in the chamber was slowly raised by circulating warm water (35 40 C) through the device. Drying was completed when the temperature in the chamber reached 36 C, and the pressure 1200 psi, ensuring that the critical point had been attained. Once drying was complete, the water was turned off and the vent valve was opened slightly to release the pressure slow ly over a co urse of 6 10 min Dr y buds were stored in a dessicator. In some cases the critical point dryer was unavailable as several parts needed to be ordered, replaced, and tested, and/or CPD failed to give good results. In these instances a chemical dry ing proce ss was substituted making use of the drying agent hexamethyldisalazane (HMDS). Buds were harvested, fixed, and dehydrated as described above. They were then immersed in HMDS (Electron Microscopy Sciences, Hatfi eld, PA, USA) for 20 min The HMDS was change d out and replaced with fresh after 20 m in and this was repeate d once more for a total of three 20 min washes. The buds were then removed from HMDS and set in open petri dishes in a dessicator, where they were allowed to air dry overnight. 2.4 Sputter Coating After drying by either method buds were dissected under a dissecting microscope such that the outer whorls of floral organs (sepals, petals, and anthers) were removed to expose the intact carpels, still attached to the receptacle (Figure 2.1) The carpels and receptacle were mounted on an SEM stub using double sided adhesive tape and the samples were
37 sputter coated with gold to prevent ch arging effects wherein static charges from the electron beam accumulate on non conductive specimens and obscu re the images (Allen, 2008) Samples were coated using an EMS 76 mini coater (Ernest F. Fullam, Inc., Schenectady, NY, USA) gold sputter coater. They were placed in a specimen chamber which was pumped down to vacuum and filled with argon gas. A current of 25 mA was appli ed for 30 s, ionizing the argon gas. Ionized atoms of gas bombard the cathode of the circuit, which is a block of gold metal, thereby dislodging gold atoms. These ions flow towards the anode and, en route, are deposited in a fine film on th e sample (Egerton, Fig. 2.1: In progress dissection of a solvent control treated bud after cr itical point drying. The pre fusion carpels are marked with C and arrows denote the region where fusion would eventually occur. After drying but prior to sputter coating, sepals ( S ), petals ( P ), and anthers ( A ) were removed with the aid of a dissecting sc ope. 40x. Figure by author.
38 2007). A total of eight 30 s bursts of voltage wer e applied giving eight coatings to the carpels. Repeated coatings ensured a continuous layer of gold for proper discharge. A colloida l silver pen was used to deposite a conductive path b etween the mounting tape and the metal surface of the SEM stub, ensuring proper grounding of the specimen. 2.5 SEM Microscopy to Asse s s Chemically treated Buds for Changes in Fusion and/or Surface Structure Carpels were visualized using a Topcon Aquila Compact SEM (Topcon Positioning Systems, Inc., Livermore, CA, USA) at the University of South Florida Department of Integrative Biology Microscopy Core Laboratory, with the assistance of Mr. Edward Haller. Additional samples were imaged using a Topcon ABT 32 Scanning Electron Microscope (Topcon Positioning Systems, Inc., Livermore, CA, USA) at New College of Florida in Sarasota, Florida. Images were captured using the Orion Digital Image Acquisition System (E.L.I. SPRL, Charleroi, Belgium). 2.6 DAB Stai ning Attempts were made to visualize patterns of ROS production at successive stages in the fusion process using the stain d iaminobenzidine (DAB). I n this study, untreated buds of various stages were harvested from whole plants grown in the growth chamber as de scribed above. They were transferred to a 12 well ceramic plate with 3 4 buds per well. Approximately 0.5 ml of 1 mg/ml DAB solution (Sigma Aldrich, St. Louis, MO, USA) in diH 2 0 was added to each well, enough to immerse the buds completely. The plat e was wrapped loosely in saran wrap and transferred to a glass cham ber where a gentle vacuum
39 was appl ied for approximately 10 min The plate was then wrapped in foil and moved to a rotating table where it was shaken at 50 rpm at room temperatu re for 24 h After the overnight ro tation, the buds were kept at 4 C while still under DAB solution for up to 48 h before being transferred to a bleaching solution of 1:1:3 (v) glycerol : acetic acid : ethanol. Incubation in this solution leached much of the chlorophy ll from the tissue without effecting the brown precipitate left by the DAB (Ramel, 2009) ; it also preserved the buds until they could be dissected under a light microscope to reveal the staining patterns of the developing carpels
40 Chapter 3: Results Treatment of cuttings with the brassinosteroid biosynthesis inhibitor Brz2001 clearly inhibited fusion, as was previously reported (Goodman, 2012; Clore et al, in revision). The overall patterns of development and the shape and ar rangement of surface cells of carpels that developed in the presence of 10 Brz2001 did not differ significantly from those of control treated samples. Treatment with the NADPH oxidase inhibitor DPI interfered with fusion in a manner distinct from that of Brz2001, with the former resulting in carpels that were partially joined but in which an apparently deep gap persisted at least part way through the tissue even after the style began to elongate and the gynoecium had achieved a height of more than 600 m (F igure s 3.1 3.2 ). Neither Brz2001 nor DPI treatment seem ed to interfere with the overall growth and development of the carpels, as gynoecia treated with either inhibitor attained a normal height and showed signs of developing elongated papilla on the la te postfusion distal tips (data not shown) The overall effects of different inhibitor treatments on carpel fusion as observed with both light microscopy and SEM throughout the course of this study are summarized in Table 3.1. Very young untreated carpels (~120 m in height) displayed what appeared to be remnants of a fine mesh covering the adaxial surface, and fibers of a similar material extending from each carpel in the direction of the other (F ig ure 3.3 ). Unfortunately, the difficulty of dissecting and drying such early stage buds without damaging the tissue made comparison of this morphology among the different treatments difficult No similar structures were observed on young carpels treated with either Brz2001 or DPI (data not
41 Fig. 3.1: Overall effects of various treatments. Gynoecia taken from cuttings grown in (L R) Brz2001, DMSO, and DPI (10 M each), then critical point dried and visualized via SEM. Brz2001 treatment clearly inhibits fusion; DPI treatment results in the persistence of a clear but not as marked fusion suture long after the carpels attain postfusion height (arrows). Each image is representative of at least three dissections.
42 Untreated c arpels were stained at various developmental stages with DAB, which produces a brown precipitate when oxidized by hydrogen peroxide thereby reporting the distribution of the latter ROS molecule (Thordal Christensen, 1997; Ramel, 2009) Distinct and reprod ucible s taining patterns were observed using this method, and these patterns changed markedly as fusion progressed (F igure 3. 4) Very young carpel primordia stain ed heavily, as did the areas between and around the emerging organs (Figure 3.4 A) More matur e carpels that were not yet pressed together stain ed faintly throughout, and heavily at the distal tips (Figure 3.4 B) Further dissection along the fusion plane of the developing gynoecium revealed that staining at this stage was concentrated in circumfer ential surface layers of the carpels and that the more central Fig. 3.2: Comparison of the persistent fusion suture (den oted by arrows) in DMSO (L) and DPI (R) treated gynoecia. The suture on the solvent treated sample is quite shallow and the underlying cells are clearly joined into a pavement like surface. The DPI treated sample, on the other hand, shows a gap along the j unction that evidently extends several cell layers deep. The gold coating may have been thicker on the left hand sample, contributing to the smoothing effect, but the cells along the fusion suture nonetheless appear to be joined to a greater extent on the sample imaged on the left.
43 Fig. 3.3: Incipient carpels from untreated buds. Arrows denote remnants of a fine mesh like covering that appears to cover the adaxial surface of the developing carpels, and similarly textured tendrils that seem to stretch towar this stage of development are larger and flatter than the surface cells of the more mature gynoecia shown in figure 3.1.
44 Fig. 3.4: DAB staining for hydrogen peroxide at various sta ges of development. Very young carpels (arrows), as well as the areas around and between them, stain reddish indicating the presence of H 2 O 2 (A). More mature carpels that have not yet made contact stain throughout, with more concentrated staining at the distal tips (B). Early fusing carpels stain more dramatically and in an area extending further down (C), while in late fusing carpels the staining pattern switches dramatically, with the top half of the gynoecium remaining unstained (D). A postfusion gyno ecium stains faintly and uniformly (E). F illustrates a cross section along the fusion plane of the same set of carpels shown in image C, demonstrating increased H 2 O 2 concentration around the outer edge (arrows), and fainter staining of the inner tissue. E ach image represents at least three dissections. A B C D E F F
45 tissue on the adaxial face, which is somewhat recessed at this early stage, stained more faintly (Figure 3.4 F). Early fusing carpels stained strongly over a region extending from the distal tips down to a region that eventually narrows to become the style (Figure 3.4 (i.e., narrow just above the developing ovaries) to form the style. At this stage of development the upper region of the gynoecium remained unstained, while the ovaries stained a faint but persistent red (Figure 3.4D) Later stage buds showed faint but apparently uniform staining over the entire gynoecial s urfaces as the style constricted further and continued to elongate (Figure 3.5 E) The correlation of distinct DAB staining patterns with apparent developme ntal stages as fusion progressed was observed consistently in buds taken at random from several pla nts (n = 5 7 for each stage of fusion) The differential staining was evident in both green buds and in those that were bleached to remove chlorophyll for clearer visualization of the DAB precipitate. Anthers, the nex t whorl of floral organs adjacent to th e carpels remained c learly unstained in all dissections as compared to developing carpels.
46 Table 3.1: Effects of Brassinosteroid Biosynthesis and NAPDH Oxidase Inhibitors o n Carpel Fusion in C. roseus Treatment Buds Treated Carpels surviving to late f usion or postfusion stage Gynoecia with abnormal fusion Summary of fusion defects Brz2001 26 14 14 Fusion significantly impaired, with two halves of gynoecium often splitting apart during the drying process. Fresh dissections are easily separated when pro bed with a needle. DPI 31 11 9 Apparently deep fusion suture is visible even on late postfusion carpels. Two halves of the gynoecium generally remain a ligned, suggesting superficial fusion. Wet dissections are easily separated and sometimes come apart wit hout probing. DMSO 17 7 1 Fusion is similar to that of gynoecia from untreated buds. A faint suture remains even on postfusion gynoecia, but extends only one or two cell layers deep Fusing and postfusing carpels resist separation by a dissecting needle.
47 Chapter 4: Discussion and Future Directions A scanning electron microscope (SEM) study was undertaken of gynoecial morphology as it changed over the course of carpel fusion in Catharanthus roseus. By performing detailed surface structure studies on carpels from cuttings that had been treated with various inhibitors, it was hoped that connections could be established work with untreated carpels (discussed in the Introduction), and the identificatio n of inhibitors that interrupted the fusion process in various ways, the investigation of the surface structure of carpels treated with these inhibitors seemed potentially fruitful I t was hypothesized that surface modifications as a result of the inhibitor s were disrupting the fusion It was additionally hy pothesized that changes in the surface structure of the carpels might occur as they prepa red to fuse or began the process. This thesis study examined normally developing carpels with the SEM and compared these to carpels that developed in the presence of Brz2001, a brassinosteroid synthesis inhibitor, and DPI, which inhibits a major source of cellular hydrogen peroxide. Neither treatment altered the surface structure of the carpels, although both appeared to interfere with fusion. SEM images of untreated, prefu sion buds verified the presence of mesh and tendril like structures on both the outer and adaxial surfaces of the carpels, The fact t hat these extra cellular structures survived the harsh chemicals and high temperatures of the critical point drying procedure, as the cuticle and other lipophilic components of the matrix evidently did not, suggests that they represent remnants of cell wal l material that extend into the
48 cuticular layer (Walker, 1975a) Cellulose microfibrils are commonly found interspersed among the waxes and cutins of the inner layers of the plant cuticle (see Figure 1.6), and cellulose fibers are known to be unaffected by critical point drying (for example Burk, 2002) The absence of these structures in more mature carpels additionally supports this hypothesis, since the cuticle of contacting epidermal cells eventually vanishes during dedifferentiation, as fusion zone cell s lose their epidermal character and become uniform parenchyma tissue (Walker, 1975b; Verbeke, 1989) Problems with this study included large amounts of time spent troubleshooting optimal procedures for preparing and imaging the plant tissues, exacerbated by the fact that repairs were needed on some key pieces of equipment Sufficiently clear and useful images were not obtained of carpels at all stages of development and for all inhibitor treatments. Better images would enable an examination of the fibrous surface structures to chart possible changes over the course of the fusion process, as well as a comparison between inhibitor treated and untreated buds. The failures of this study led to the determination that a very gradual increase in d rying chamber temperature is the most important factor in preserving delicate structures for SEM. Allowing adequate time for the tissue to become infiltrated with CO 2 is also important, bearing in mind that diffusion will be slower through the cell wall an d that most protocols are written with animal tissue in mind. Future studies could m ake use of this information and it is hoped, obtain better results. N o apparent differences in the shape and arrangement of surface cells between control carpels and those treated with inhibitors that prevent complete fusion were found in this study This paucity of results lends support to the hypothesis that changes in the
49 structure and properties of the extracellular matrix, rather than changes in the cells themselves, initially regulate the fusion process. Ch anges i n cuticle permeabi lity con current with the fusion process, identification of cuticle synthesis mutants that manifest an abnorm al organ fusion phenotype in Arabidopsis and increased efficiency of pathogen recognition signaling in plants wit h cuticle mutations, have all been reported Collectively, these findings point to a model of adjacent epidermal cell signaling in which the cuticle is a key regulator and where modulating cuticle properties may be an important signal ing mechanism. (These studies are reviewed and discussed in greater deta il in the Introduction, and some of the ideas linking various lines of evidence we re originally proposed by Lolle ( 1999 ) ). The current study shows that inhibitors of fusion do not nec essarily alter the surface structures of the fusing carpels. Continuation of the SEM work to further examine the fibrous extracellular structures observed on some samples would be one way to explore cuticle structure. The cuticle proper is not well preserv ed during the critical point drying process, but the fibrous cell wall extensions that remain on the surface of the carpels are well preserved and may provide clues as to the nature of the cuticle at various stages. Other possibilities, perhaps better suit ed to investigating the role of the cuticle in carpel fusion include the use of dyes and stains to attempt to track various cuticle components of the cuticle throughout the fusion process. L ipophilic dyes such as Nile red or Sudan black, or pectin stains such as Alcian blue or ruthenium red for pectins, might be good candidates. Cryo SEM is a modified SEM technique which makes use of extensive cooling equipment at both the coating and the imaging stages of the microscopy process, such that samples can be k ept
50 slush, rather than being subjected to critical point drying. This technique provide s exquisitely detailed images s urface wax structure on e pidermal tissue (see for example Pighin, 2004), and could likely be very useful in deciphering changes in cuticle structure and their relationship to increased permeability. Several parts of this thesis work dealt with a potential role for reactive oxygen species signaling d uring C. roseus carpel fusion. Treatment of cuttings with DPI resulted in a persistent fusion suture that was different in character from that observed with either Brz2001 or the DMSO control (Figures 3.1 and 3.2). Additionally, staining with DAB revealed distinc t and dynamic patterns of hydrogen peroxide distribution over the course of fusion (Figure 3.4). Taken together, these results re present preliminary evidence that suggests ROS signaling is important in this process. The patterns of ROS distribution revealed by DAB staining in this study resist easy characterization. Hydrogen peroxide levels are markedly increased (as compared to surrounding tissue) when very young carpel primordia are first forming, suggesting that hydrogen peroxide production and/or accumul ation may correlate with early carpel organogenesis As carpels matured, DAB staining occurred in areas that correspond to increased cuticle permeability (as demonstrated with TBO assays by Goodman, 2012), and the increased levels of hydrogen peroxide wer e most marked in the outer layers of tissue. DAB staining was restricted longitudinally to the region of the carpels that ultimately fuse (i.e. the future stigma, style, and distal ovaries) although it occurred circumferentially around the carpels and was not restricted to contacting surfaces. ROS levels at this stage are increased in areas that are competent to fuse, but the increase is not dependent on contact of opposing carpels nor restricted to the fusion zone along the
51 adaxial faces A possible exp lanation for this would be that ROS are involved in increasing cuticle permeability that subsequently allows for the exchange of fusion factors. The existence of cell wall specific H 2 O 2 producing peroxidases (Liszkay, 2003) and documented role of ROS in loosening cell walls to allow for elongation during vegetative growth (Muller, 2009) c ould be construed to support to this hypothesis. The dramatic switch in ROS patterning that was observed in this study during the la te postfustion stage suggests multiple roles for R OS that depend on other factors O nce at least surface level fusion is complete, hydrogen peroxide appears to become more important in other processes (than carpel fusion), perhaps i ncluding ovary development. It is unlikely that the ROS patterns observed can be attributed solely to elongation growth, as if this were the case heavy staining would be expected in the area of the future style, where the most significant elongation occurs. Such staining patterns are not observed. Likewise, the production of hydrogen peroxide would eventually be expected in the future stigma region in preparation for pollen capture ( McInnis, 2006). In fact, however, the future stigma region is conspicuously unstained for a tim e during the late fusing stage and in postfusion gynoecia is no more heavily stained than surroundi ng tissue. This suggests that the patterns of DAB staining are not entirely explainable by hydrogen peroxide acting in its well established roles in the aforementioned processes, and could potentially suggest a fusion specific role for this ROS in carpel d evelopment in this system. This thesis study tentatively adds hydrogen peroxide to a list of factors that have been shown previously to play a role in the postgenital carpel fusion of C. roseus namely brassinosteroid hormones and increased cuticle perme ability. The current study seems to
52 also indicate that overt changes in surface structure of the carpels are not a means by which the inhibitors Brz2001 and DPI interfere with carpel fusion. The obvious larger question that remains to be answere d is how th ese signature pathways (and, more than likely, other important players as yet unidentified) work together to regulate the carpel fusion process. Recent work that may help illuminate these mechanisms centers around a family of genes known as LATERAL ORGAN B OUNDARIES (LOB). LATERAL ORGAN BOUNDARIES (Shuai, 2002) represent a family of genes identified in Arabidopsis whose protein products share a conserved domain known as LOB LOB proteins are transcription factors involved in maintaining the integrity of edge s and clean separation of organs that develop from the meristem (Shuai, 2002). Recently the LOB family was shown to negatively regulate brassinosteroid accumulation at organ boundaries (for example, between axillary stems and cauline leaves), via transcri ptional activation of the brassinosteroid inactivating enzyme BAS 1 (Bell, 2012). Ectopic LOB expression down regulates brassinosteroid responses, whereas loss of function LOB mutants show abnormal organ fusions. LOB expression is in turn regulated by bras sinosteroids (Bell, 2012), suggesting complex feedback loops that help to regulate the process of organ separation. These findings provide a transcriptional link between brassinosteroid action and organ fusions in Arabidopsis one which may be at play in carpel fusion in C. roseus Furthermore, microarray experiments identified several genes responsive to the LOB transcription factors that have a role in modifying both cell wall structure and respon s e s to pathogen invasion (Bell, 2012). Both of these alter ations could coincide with modifications to the cuticle and the associated permeability in C. roseus although more
53 research would surely be needed to confirm this speculation. Finally, several LOB transcription factors have been shown to be up regulated i n response to hypoxic stress (Liu, 2005), suggesting that LOB expression may be responsive to changes in ROS levels. Lateral Organ Boundaries thus represent one potential link between a diverse set of processes and signaling mechanisms that appear to play a role in carpel fusion in C. roseus. Much further study would be needed to begin to examine this hypothesis, but the results of this thesis project together with previously published data derived from studies on both C. roseus and Arabidopsis suggest that it would be an interesting and potentially useful avenue of inquiry. As a conclusion to this thesis study, several ideas present themselves f or further investigation of this system. A stable transformation of C. roseus with a GUS gene reporter system w as recently reported (Wang 2012). This elegant technique for visualizing spatial and temporal patterns of expression could be useful here once candidate genes are identified, especially given that the small size of the carpels has made traditio nal methods of extracting and analyzing DNA very difficult in the past. Another technology which should be available at New College in the near future, is known as Solid Phase Microextraction, o r SPME. The use of a mi crofiber capillary needle allows for the extraction, and subsequent GC/MS analysis, of miniscule volumes of volatile emissions or exudates. This technique has been used to ex tract brassinolide from pollen samples ( Pan, 2012). It wou ld likely corroborate the i m portance of bra ssinosteroid hormones in the chemical exchange that mediates C. roseus carpel fusion, a nd could additionally identify as yet unknown components. Finally, f luorescent hydrogen peroxide detectors might help to characterize the distribution patterns hinted at by DAB
54 staining in this st udy. Assays for other ROS would also be beneficial, particularly those such as nitric oxide that are kn own to interact with hydrogen peroxide in an antagonistic manner ( i.e. Krasniewski, 2013).
55 R EFERENCES : Allen, Terrence, ed. Introduction to Electron Microscopy for Biologists. Burlington, MA: Academic, 2008. Apel, K., and Hirt, H. (2004). Reactive oxygen s pecies: metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 55: 373 399. Arber, E. A .N., and Parkin, J. (1907). On the origin of Angiosperms. J. Linn. Soc. Bot. 38: 28 90. Armbruster, W.S., Debevec, E.M., and Wilson, M.F. (2002 ). Evolution of syncarpy in angiosperms: theoretical and phylogenetic analyses of the effects of carpel fusion on offspring quality. J. Evol. Bio l 15: 657 672. Bai, X., Todd, C.D., Desika, R., Yang, Y., and Hu, X. (2012). N 3 oxy decanoyl L homoserine la ctone activates auxin induced adventitions root formation via hydrogen peroxide and nitric oxide dependent cyclic GMP signaling in Mung Bean. Plant physiol. 158: 725 736. Balusk a, F., Mancuso, S., Volkmann, D., and Barlow, P.W. (2010). Root apex transitio n zone: a signaling response nexus in the root. Trends Plant Sci. 15: 402 408. Bell, E.M., Lin, W., Husbands, A.Y., Yu, L., Jagantha, V., Jablonska, B., Mangeon, A., Neff, M., Girke, T., and Springer, P.S. (2012). Arabidopsis LATERAL ORGAN BOUNDARIES nega tively regulates brassinosteroid accumulation to limit growth in organ boundaries. Proc. Natl. Acad. Sci. 109: 21146 21151. Bessire, M., Chassot, C., Jacquat, A.C., Humphry, M., Borel, S., MacDonald Comber, J., Metraux, J.P., and Nawrath, C. (2007). A pe rmeable cuticle in Arabidopsis leads to a strong resistance to Botrytis cinerea EMBO J. 26: 2158 2168. Bhattacharjee, S. (2012). The language of reactive oxygen species signaling in p lants. J. Bot. 2012: Article ID 985298, 22 pages. doi:10.1155/2012/985 298 Bolwell, G.P., and Wojtaszek, P. (1997). Mechanisms for the generation of reactiv e oxygen species in plant defens e a broad perspective. Physiol. Mol. Plant Path. 51: 347 366. Boke, N.H. (1949). Development of the stamens and carpels in Vinca rosea L. Amer. J. Bot. 36: 535 547.
56 Boonsirichai, K., Guan, C., Chen, R., and Masson, P.H. (2002). Root gravitropism: an experimental tool to investigate basic cellular and molecular processes underlying mechanosensing and signal transmission in plants. Annu. R ev. Plant Biol. 53: 421 447. Bourdenx, B., Bernard, A., Domergue, F., Pascal, S., Leger, A., Roby, D., Pervent, M., Vile, D., Haslam, R.P., Napier, J.A., Lissire, R., and Joubes, J. (2011). Overexpression of Arabidopsis ECERIFERUM1 promotes wax very long chain alkane biosynthesis and influences plant response to biotic and abiotic stresses. Plant Phys iol 156: 29 45. Buchholz, A. (2006). Characterization of the diffusion of non electrolytes across plant cuticles: properties of the lipophilic pathway. J E xp. Biol. 57: 2501 2513. Burk, D.H., and Ye, Zheng Hua (2002)). Alteration of oriented deposition of cellulose microfibrils by mutation of a katanin like microtubule severing protein. Plant Cell 14: 2145 2160. Carland, F. Fujioka, S., and Nelson, T. (201 0). The sterol methyltrasnferases SMT1, SMT2, and SMT3 incluence Arabidopsis development through nonsteroid products. Plant Physiol. 153: 741 756. Clore, A.M. (2013). Cereal grass pulvini: Agronomically significant models for studying gravitropism signali ng and tissue polarity. Am. J. Bot. 100: 101 10. Clore, A. M., Durbak, A.R., Goodman, K.J., Tatum, M.A., and Kahn, B.B. (In revision). Roles for brassinosteroid signaling and changes in cuticle permeability in carpel fusion during Catharanthus roseus gyno ecium development. Clore, A.M., Doore, S.M., and Tinnirello, S.M.N. (2008). Increased levels of reactive oxygen species and expression of a cytoplasmic aconitase/iron regulatory protein 1 homolog during the early response of maize pulvini to gravistimula tion. Plant, Cell, and Env. 31: 144 158. Clouse, S.D., Langford, M., and McMorris, T.C. (1996). A brassinosteroid insensitve mutant in Arabidopsis thaliana exhibits multiple defects in growth and development. Plant Physiol. 111: 671 678. Clouse, S.D. (20 11). Brassinosteroid signal transduction: from receptor kinase activation to transcriptional networks regulatin g plant development. Plant Cell 23: 1219 1230.
57 Curvers, K., Seifi, H., Moulle, G., De Rycke, R., Asselbergh, B., Van Hecke, A., Vanderschaeghe, D., Hofte, H.R., Callewaert, N., Van Breusegemm, F., and Hofte, M. M. (2010). ABA deficiency causes changes in cuticle permeability and pectin composition that influence tomato resistance to Bo trytis cinerea Plant Physiol. 154: 847 860. Deter, R.A., Oh, M.H., Larue, C.T., and Huber, S.C. (2011). Crosstalk between brassinosteroids and ROS signaling via oxidation of methionine residues in the extracellular domain of the BRI1 receptor k inase. American Society of Plant Biologists meeting abstracts, Abstract # P20033. Accessed 3 27 203. Dornelas, M.C., van Lammeren, A.A.M., and Kreis, M. (2000). Arabidopsis thaliana SHAGGY related protein kinases (AtSK11 and 12) function in perianth and gynoecium development. Plant J. 21: 419 429. Doke, N., Miura, Y., Sanchez L.M., Park, H J., Noritake, T., Yoshioka, H., and Wakakita, K. (1996). The oxidative burst protects plants against pathogen attack: mechanism and role as an emergency signal for plant bio defense a review. Gene 179: 45 51. Edlund, A.F., Swanson, R., a nd Preuss, D. (4004). Pollen and stigma structure and function: The role of diversity in pollination. Plant Cell 16: S84 S97. Egerton, Ray. Physical Principles of Electron Microscopy. New York: Springer, 2007. Electron Microscopy Sciences (2013). Technica l Data Sheets: Critical point drying principals. http://www.emsdiasum.com/microscopy/technical/datasheet/critical_drying.aspx Accessed 1/21/2013. Endress, P.K. ( 1982). Syncarpy and alternative modes of escaping disadvantages of apocarpy in primitive angiosperms. Taxon 31: 48 52. Endress, P.K (2006). Angiosperm floral evolution: Morphological development framework. Advances in Botanical Research: Incorporating A dvances in Plant Pathology. 44: 1 61. Endress, P.K. and Igersheim, A. (2002). Gynoecium structure and evolution in basal angiosperms. Int. J. Plant Sci. 161: S211 S223. Finkel, T (2011). Signal transduction by reactive oxygen species. J. Cell Biol. 194: 7 75.
58 Foyer, C.H., and Noctor, G. (2005). Redox homeostasis and antioxidant signaling: a metabolic interface between stress perception and physiological responses. The Plant Cell 17: 1866 1875. Foyer, C.H., and Noctor, G. (2011). Ascorbate and glutathi one: the heart of the redox hub. Plant Physiol. 155: 2 18. Fujino, G., Noguchi, T., Takeda, K., and Ichijo, H. (2006). Thioredoxin and protein kinases in redox signaling. Semin. Cancer Biol. 16: 427 435. Fukuda, H. (1997). Tracheary element differentia tion. The Plant Cell 9: 1147 1156. Gampala, S.S., Kim, T.W., He, J.X., Tang, W., Deng, Z., Bai, M., Guan, S., Lalonde, S., Sun, Y., Gendron, J.M., Chen, H., Shibagaki, N., Ferl, R.J., Ehrhardt, D., Chong, K., Burlingame, A., and Wang, Z.Y. (2007). An esse ntial role for 14 3 3 proteins in brassinosteroid signal transduction in Arabidopsis Dev Cell 13: 177 189. Goodman, K.J. (2012). Evidence for brassinosteroid signaling and changes in cuticle permeability during carpel fusion in Catharanthus roseus. Under graduate thesis. New College of Florida. Sarasota, FL, USA. Greebe, M. (2004). Ups and downs of tissue and planar polarity in plants. BioEssays 26: 710 729. Groves, M.D., Spencer, G.F., Rowhedder, W.K., Mandava, N., Worley, J.F., Warthen, J.D., Steffens, G.L., Flippen Anderson, J.L., and Cook, J.C. (1979.) Brassinolide, a plant growth promoting steroid isolated from Brassica napus pollen. Nature. 281: 216 217. Gupta, K.J., Shah, J.K., Brotman, Y., Jahnke, K., Willmitzer, L., Kaiser, W.M., Bauwe, H., and Igamberdiev, A.U. (2012). Inhibition of aconitase by nitric oxide leads to induction of the alternative oxidase and a shift of metabolism towards biosynthesis of amino acids. J. Exp. Bot. 63: 1773 1784. Hardin, S.C., Larue, C.T., Oh, M.H., Jain, V., and H uber, S.C. (2009). Coupling oxidative signals to protein phosphorylation via methionine oxidation in Arabidopsis Biochem J. 422: 305 312. Hatwig, T., Chuck, G.S., Fujioka, S., Klempien, A., Weizbaurer, R., Potluri, D.P., Choe, S., Johal, G.S., and Schulz B. (2001). Brassinosteroid control of sex determination in maize. P roc. N atl. A cad. S ci 180: 19814 19819.
59 He, P., Shan, L., and Sheen, J. (2007). Elicitation and suppression of microbe associated molecular pattern triggered immunity in plant microbe in teractions. Cell. Microbiol. 9: 1385 1396. Hiscock, S.J., Bright, J., McInnis, S.M., Desikan, R., and Hancock, J.T. (2007). Signaling on the stigma: potential new roles for ROS and NO in plant cell signaling. Plant Sig. and Behav. 2: 21 24. Jallais, Y. Hothorn, M., Belkhadir, Y., Dabi, T., Nimchuck, Z.L., Meyerowitz, E.M., and Chory, J. (2011 ) Tyrosine phosphorylation controls receptor activation by triggering membrane release of its kinase inhibitor. Gene Dev. 25: 232 237. Jiang, M., and Zhang, J. ( 2002). Water stress induced abscisic acid accumulation triggers the increased generation of reactive oxygen species and up regulates the activities of antioxidant enzymes in maize leaves. J. Exp. Bot. 53: 2401 2410. Joo, J.H., Bae, Y.S., and Lee, J.S. (20 01). Role of auxin induced reactive oxygen species in root gravitropism. Plant Physiol. 126: 1055 1060. Kim, T.W., Guan, S., Sun, Y., Deng, Z., Tang, W., Shang, J.X., Sun, Y., Burlingame, L., and Wang, Z.Y. (2009). Brassinosteroid signal transduction from cell surface receptor kinases to nuclear transcription factors. Nature Cell Biol. 11: 1254 1260. Kim, T.W. and Wang, Z.Y. B (2010). Brassinosteroid signal transduction from receptor kinases to transcription factors. Annu. Rev. Plant Biol. 61: 681 704. K rolikowski, K. A., Victor, J.L., Wagler, T.N., Lolle, S.J., and Pruitt, R.E. (2003). Isolation and characterization of the Arabidopsis organ fusion gene HOTHEAD Plant J. 35: 501 511. Kuppusamy, K.T., Chen, A.Y., and Nemhauser, J.L. (2009). Steroids are r equired for epidermal cell fate establishment in Arabidopsis roots. P roc. N atl. A cad. S ci. 106: 8073 8076. Kurdyokov, S., Faust, A., Trenkamp, S., Bar, S., Franke, R., Efremova, N., Tietjen, K., Schrieber, L., Saedler, H., and Yephremov, A. (2006). Geneti c and biochemical evidence for involvement of HOTHEAD in the biosynthesis of long dicarboxylic fatty acids and formation of extracellular matrix. Planta 224: 315 329. Kwasniewski, M., Chwialsowska, K., Kwasniewska, J., Kusak, J., Siwinski, K., Sszarejko, I. (2013). Accumulation of peroxidase relat ed oxygen species in trichoblasts correlates with root hair initiation in barley. J. Plant Physiol 170: 1 195.
60 Larkin, J.C., Brown, M.L., and Schiefelbein, J. (2003). How d o cells know what they want to be when they grow up? Lessons from epidermal patte rning in Arabidopsis Annu. Rev. Plant Biol. 54: 403 430. Leslie, A.B., and Boyce, K.C. (2012). Ovule function a nd the evolution of angiosperm reproductive innovations. Int. J. Plant Sci. 173: 640 648. Bard, A., Binda, M., Serrano, M., Abou Mansour, E., Balet, F., Schoonbeek, H.J., Hess, S., Mir, R., Leon, J., Lamotte, O., and Metraux, J.P. (2001). A permeable cuticle is associated with the release of reactive oxygen species and induction of innate immunity. PLoS Path. 7: e1002148, p. 1 17. Li, J., and Chory, J. (1997). A putative leucine rich repeat receptor kinase involved in brassinosteroid signal transduction. Cell 90: 929 938. Li, J., and Nam, K.H. (2002). Regulation of brassinosteroid signaling by a GSK3/SHAGGY like kinase. S cience 295: 1299 1301. Li, S.W., Xue, L., Xu, S., Feng, H., and Lizhe, A. (2009). Hydrogen peroxide acts as a signal molecule in the adventitious root formation of mung bean seedlings. Environ. Exp. Bot. 65: 63 71. Liszkay, A., Kenk, B., and Schopfer, P. (2003). Evidence for the involvement of cell wall peroxidase in the generation of hydroxyl radicals mediating extension growth. Planta 4: 658 667. Liu, F., Vantoai, T., Moy, L.P., Bock, G., Linford, L.D., and Quackenbush, G. (2005). Global transcription profiling reveals comprehensive insights into hypoxic response in Arabidopsis. Plant Physiol. 137: 1115 1129. Lloyd, D.G., and Wells, M.S. (1992). Reproductive biology of a primitive angiosperm, Pseudowintera colorata (Winteraceae), and the evolution of p ollination systems in the Anthophyta. Plant Syst. Evol. 181: 77 95. Lolle, S.J., Cheung, A.Y., and Sussex, I.M. (1992). Fiddlehead : an Arabidopsis mutant constitutively expressing an organ fusion program that involves interactions between epidermal cells. Dev. Biol. 152: 383 392. Lolle, S.J., Berlyn, G.P., Engstrom, E.M., Krolikowski, K.A., Reiter, W.D., and Pruitt, R.E. (1997). Developmental regulation of cell interactions in the Arabidopsis fiddlehead 1 mutant: A role for the epidermal cell wall and cut icle. Dev. Biol. 189: 311 321.
61 Lolle, S.J. and Pruitt, R.E. (1999). Epidermal cell interactions: a case for local talk. Trends in Plant Sci. 4: 14 20. Mandava, N.B. (1988). Plant growth promoting brassinoster oids. Annu.Rev. Plant Physiol. Plant Mol. Bio l. 39: 23 52. Marondedze, C., Turek, K., Parrott, B., Thomas, L., Jankovic, B., Lilley, K., and Gehring, C. (2013). Structural and functional characteristics of cGMP dependent methionine oxidation in Arabidopsis thaliana proteins. Cell Comm. Sig. 11: 1 7. McInnis, S.M., Desikan, R., Hancock, J.T., and Hiscock, S.J. (2006). Production of reactive oxygen species and reactive nitrogen species by angiosperm stigmas and pollen: potential signaling crosstalk? New Phytol. 172: 221 228. Meng, L., Wong, J.H., Fel dman, L.J., Lemaux, P.G., and Buchanan, B.B. (2010 ). A membrane associated thioredoxin required for plant growth moves from cell to cell, suggestive of a role in intercellular communication. P roc. N atl. A cad. S ci. 107 : 3900 390 5. Mittler, R., Vanderauwera S., Suzuki, N., Miller, G., Tognetti, V.B., Vandepoele, K., Gollery, M., Sulaev, V., and Van Breusegem, F. (2011). ROS signaling: the new wave? Trends Plant Sci. 16: 300 309. Monshausen, G.B., Bibikova, T.N., Messerli, M.A., Shi, C., and Gilroy, S. (20 07). Oscillations in extracellular pH and reactive oxygen species modulate tip growth of Arabidopsis root hairs. Proc. Natl. Acad. Sci. 104: 20996 1001. Muller, K., Linkies, A., Vreeburg, R.A.M., Fry, S.C., Krieger Liszkay, A., and Leubner Metzger, G. (20 09). In vivo cell wall loosening by hydroxyl radicals during cress seed germination and elongation growth. Nakashita, H ., Yasuda, M ., Nitta, T ., Asami, T ., Fujioka, S ., Arai, Y ., Sekimata, K ., Takatsuto, S ., Yamaguchi, I ., and Yoshida, S (2003). Brassinost e roid functions in a broad range of disease resistance in t obacco and rice. Plant Journal 33: 887 898 Navarre, D.A., Wendehene, D., Durne r, J., Noad, R., and Klessig, D.F., (2000). Nitric oxide modulates the activity of tobacco aconitase. Plant Phsiol. 122: 5 73 582. Nawrath, C. (2006). Unraveling the complex network of cuticular structure and function. Curr. Opin. Plant. Biol. 9: 281 287. Nicaise, V., Roux, M., and Zipfel, C. (2009). Recent advances in PAMP triggered immunity against bacteria: pattern recognition receptors watch over and raise the alarm. Plant Physiol. 150: 1638 1657.
62 Nomura. T., Uneo, M., Yamada, Y., Takatsuto, S., Tak euchi, Y., and Yokota, T. (2007). Roles o f brassinosteroids and related mRNAs in p ea seed growth and germination. Plant Physiol. 143: 1680 1688. (1964). Polychromatic staining of plant cell walls by toluidine b lue O. Protoplasma 59: 368 373. Pan, J., Hu, Y., Liang, T., and Li, G. (2012). Preparation of solid phase microextraction fibers by in mold coating strategy for derivatization analysis of 24 epibrass inolide in pollen samples. J. Chromatog. A 1262: 29 55. Pighin, J.A., Zheng, H., Balakshin, L., Goodman, I., Western, T., Jetter, R., Kunst, L., and Samuels, A.L. (2004). Plant cuticular lipid export requires an ABC transporter. Pruitt, R. E., Vielle Calzada, J.P., Ploense, S.E. Grossniklaus, U., and Lolle, S.J. (2000). FIDDLEHEAD a gene required to suppress epidermal cell interactions in Arabidopsis encodes a putative lipid biosynthetic enzyme. P roc N at l A cad. S ci. 97: 1311 1316. Qin, W.M., Lan, W.Z., and Yang, X. ( 2004). Involvement of NADPH oxidase in hydrogen peroxide accumulation by Aspergillus niger elicitor induced Taxus chinensis c ell cultures. J. Plant Physiol. 161: 355 361. Ramel, F., Sulmon, C., Bogard, M., Vouee, I., and Gouesbet, G. (2009). Differential patter ns of reactive oxygen species and antioxidative mechanisms during atrazine injury and sucrose induced tolerance in Arabidopsis thaliana plantlets. BMC Plant Biol. 9; http://www.biomedcentral.com/14 71 2229/9/28 Russinova, E., Borst, J.W., Kwaaitaal, M., Cano Delgado, A., Yin, Y., Chory, J., and de Vries, S.C. (2004). Heterodimerization and e ndocytosis of Arabidopsis brassinosteroid r ecepto rs BRI1 and AtSERK3 (BAK1). Plant Cell 16: 3216 3229. Ryu, H., Cho, H., Kim, K., and Hwang, I. (2010). Phosphorylation dependent nucleocytoplas mic shuttling of BES1 is a key regulatory event in brassinosteroid signaling. Mol. Cells. 29: 283 290. Saidi, Y., Hearn, T.J., and Coates, J.C. (2012). Function and evolut GSK3/Shaggy like kinases. Trends Plant Sci 17: 39 46. Samuels, L., Kunst, L., and Jetter, R. (2008). Sealing plant surfaces: Cuticular wax formation by epidermal cells. Annu. Rev. Plant Biol. 59: 638 707 Savaldi Goldstein, S., Peto, C., a nd Chory, J. (2007). The epidermis both drives and restricts plant shoot growth. Nature 446: 199 202.
63 Schaller, H. (2004). New aspects of sterol biosynthesis in growth and development of higher plants. Plant Physio l Biochem. 42: 465 476. Schreiber, L. ( 2005). Polar paths of diffusion across plant cuticles: new evidence for an old hypothesis. Ann Bot London 95: 1069 1073. Sekimata, K., Kimura, T., Kaneko, I., Nakano, T., Yoneyama, T., Takeuchi, Y., Yoshida, S., and Asami, T. (2001). A specific brass inosteroid biosynthesis inhibitor, Brz2001: evaluation of its effects on Arabidopsis c ress, tobacco, and rice. Planta 213: 716 721. She, J., Han, Z., Kim, T.W., Wang, J., Cheng, W., Chang, J., Shi, S., Wang, J., Yang, M., Wang, Z. Y., and Chai, J. (2011) Structural insight into brassinosteroid per ceptio n by BRI1. Nature 474: 472 477. Shiu, S.H., Karlowski, W.M., Pan, R., Tzeng, Y.H., Mayer, K.F., and Li, W.H. (2004). Comparative analysis of the receptor like kinase family in Arabidopsis and rice. Plant Cell 16: 1220 1234. Shuai, B., Reynaga Pena, C.G., and Springer, P.S. (2002). The Lateral Organ Boundaries gene defines a novel, plant specific gene family. Plant Physiol. 179: 747 761. Siegal, B.A. and Verbeke, J.A. (1992). Diffusible factor s essential for epidermal cell redifferentiation in Catharanthus roseus. Science 244: 580 582. Smirnova, A., Leide, J., and Riederer, R. (2013). Deficiency in a very long chain fatty acid beta ketoacyl coenzyme A synthase of tomato impairs microgametogenesis and c aus es floral organ fusion. Plant Phys iol 161: 196 209. Smyth, D.R., Bowman, J.L., and Meyerowitz, E.M. (1990). Early flower development in Arabidopsis. Plant Cell 2: 755 767. Soltis, P.S., and Soltis, D.E. (2004). The origin and diversification of angiosp erms. Am. J. Bot. 91: 1614 1626. Stebbins, G.L. (1974). Flowering Plants: Evolution Above the Species Level. Harvard University Pr ess, Cambridge, MA, USA. Swanson, R., Edlund, A.F., and Preuss, D. (2004). Speci es specificity in pollen pistil interactions Annu. Rev. Genet. 38: 793 818. Tanaka, T., Tanaka, H., Machida, C., Watanabe, M., and Machida, Y. (2004). A new method for rapid visualization of defects in leaf cuticle reveals five intrinsic patterns of surface defects in Arabidopsis Plant J. 37: 139 146.
64 Tang, W., Kim, T.W., Oses Prieto, J.A., Sun, Y., Deng, Z., Zhu, S., Wang, R., Burlingame, A., and Wang, Z.Y. (2008). BSKs mediate signal transduction from the receptor kinase BRI1 in Arabidopsis. Science 321: 557 560. Taylor, D.W., and Hickey, L.J. (1996). Flowering plant origin, evolution and phylogeny, pp 1 3. Chapman and Hall, New York. The Arabidopsis Genome Initiative (2000). Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408: 796 815. Tsukagoshi, H., Bus ch, W., and Benfey, P.N. (2010). Transcriptional regulation of ROS controls transition from proliferation to differentiation in the root. Cell 143: 606 616. Van Der Heijden, R., Jacobs, D.E., Snoeijer, W., Hallard, D., and Verpoorte, R. (2004). The Cathar anthus alkaloids: pharmacognosy and biotechnology. Curr. Med. Chem. 11: 607 28. Van Norman, J.M., Breakfield, N.W., and Benfey, P.N. (2011). Intercellular communicatio n during plant development. Plant Cell 23: 855 864. Verbeke, J.A. (1989). Stereologica l analysis of ultrastructural changes during induced epidermal cell redifferentiation in developing flowers of Catharanthus roseus (Apocynaceae ). Amer. J. Bot. 76: 952 957. Verbeke, J.A. (1992). Fusion events during floral morphogenesis. Annu. Rev. Plant Physiol. Plant Mol. Biol. 43: 583 598. Verbeke, J.A. and Walker, D.B. (1986). Morphogenetic factors controlling differentiation and dedifferentiation of epidermal cells in the gynoecium of Catharanthus roseus II. Diffusible morphogens. Planta 168: 43 49. Vert, G. and Chory, J. (2006). Downstream nuclear events in brassinosteroid signaling. Nature 441: 96 100. Vianello, A., Macri, F. (1991). Generation of superoxide anion and hydrogen peroxide at the surface of plant cells. J. Bioenerg. Biomembr. 23: 409 423. Wada, K., Kondo, H., and Marumo, S. (1985). A simple bioassay for brassinosteroids: A wheat leaf unrolling test. Agric. Biol. Chem. 49: 2249 2251.
65 Walker, D.B. (1975a). Postgenital carpel fusion in Catharanthus roseus (Apocynaceae). I. Light and sc anning electron microscopic study of gynoecial ontogeny. Amer. J. Bot. 62: 457 467. Walker, D.B. (1975b). Postgenital carpel fusion in Catharanthus roseus III. Fine structure of the epidermis during and after fusion. Protoplasma 86: 43 63. Walker, D.B. ( 1978). Morphogenetic factors controlling differentiation and dedifferentiation of epidermal cells in the gynoecium of Catharanthus roseus I. The role of pressure and cell confinement. Planta 142: 181 186. Walsh, N.E., and Charlseworth, D. (1992). Evoluti onary interpretations of differences in pollen tube growth rates. Q. Rev. Biol. 67: 19 37. Wang, Q., Xing, S., Pan, Q., Yuan, F., Zhao, J., Tian, Y., Chen, Y., Wang, G., and Tang, K. (2012). Development of efficient Catharanthus roseus regeneration and trans formation system using agrobacterium tumefaciens and hypocotyls as explants. BMC Biotech. 12: 34. Wang, X.F., Armbruster, W.S., and Huang, S.Q. (2011). Extra gynoecial pollen tube growth in apocarpous angiosperms is phylogenetically widespread and probably adapt ive. New Phytologist 193: 253 260. Wang, Z.Y., Xiong, L., Li, W., Zhu, J.K., and Zhu, J. (2011). The plant cuticle is required for osmotic stress regulation of abscisic acid biosynthesis and osmotic stress tolerance in Arabidopsis Plant Cell 23: 1971 198 4. Wang, X., Kota, U., He, K., Blackburn, K., Li, J., Goshe, M.B., Huber, S.C., and Clouse, S.D. (2008). Sequential transphosphorylation of the BRI1.BAK1 receptor kinase complex imacts early events in brassinosteroid signaling. Dev. Cell 15: 220 235. Wil lemsen, V., Friml, J., Grebe, M., van den Toorn, A., Palme, K., and Scheres, B. Cell polarity in PIN protein positioning in Arabidopsis require STEROL METHYLTRANSFERASE1 function. Plant Cell 15: 612 625. Williams J.H. (2012). Pollen tube growth rates and the diversification of flowering plant cycles. Int. J. Plant Sci. 173: 649 661. Xia, X.J., Wang, Y.J., Zhou, Y.H., Tao, Y., Mao, W.H., Shi, K., Asami, T., Chen, Z., and Yu, J.Q. (2009). Reactive oxygen species are involved in brassinosteroid induced str ess tolerance in cucumber. Plant Physiol. 150: 801 814.
66 Xiao, F., Goodwin, S.M., Xiao, Y., Sun, Z., Baker, D., Tang, X., Jenks, M.A., and Zhou, J.M. (2004). Arabidopsis CYP86A2 represses Pseudomonas syringae type III genes and is required for cuticle deve lopment. Embo J. 23: 2903 2913. Yamamoto, R., Fujioka, S., Demura, T., Takatsuto, S., Yoshida, S., and Fukoda, H. (2001). Brassinosteroid levels increase drastically prior to morphogenesis of tracheary elements. Plant Phys iol 125: 556 563. Yang, C. J., Z hang, C., Lu, Y.N., Jin, J.Q., and Wang, X.L. (2011). The mechanisms of Molecular Plant. 4: 588 600. Yeats, T.H., Buda, G.J., Wang, Z., Chehanovsky, N., Moyle, L.C., Jetter, R., Scha ffer, A.A., and Rose, J.K. (2012). The fruit cuticles of wild tomato species exhibit architectural and chemical diversity, providing a new model for studying the evolution of cuticle function. Plant Cell 35: 1971 1984. Zechmann, B., Koffler, B.E., and Rus sell, S.D. (2011). Glutathione synthesis is essential for pollen g ermination in vitro BMC Plant Biol. 11: 54. Zinkl, G.M., Zwiebel, B.I., Grier, D.G., and Preuss, D. (1999). Pollen stigma adhesion in Arabidopsis: a species specific interaction mediate d b y lipophilic molecules in the pollen exine. Develop ment 126: 5431 5440.