ABSTRACT
A MORPHOLOGICAL AND ANATOMICAL INVESTIGATION OF SHOOT APICAL MERISTEMS EXPRESSING RING FASCIATION IN CLARKIA TEMBLORIENSIS
by Kilian TysonMayer
Fasciation is a growth abnormality in shoot and root meristems of many vascular plants which leads to the development of enlarged, supernumerary, and misshapen stems, leaves, floral organs, and fruits. Artificial selection for modified phenotypes has occurred since the dawn of agriculture and is responsible for many commercially available fruits and vegetables today. Clarkia tembloriensis is a California wildflower expressing fasciation in certain populations when grown under abnormal environmental conditions in a laboratory setting. This makes it an excellent model organism to study the effect of abnormal environmental conditions on the expression of fasciation. In this investigation, shoot apex morphology and anatomy were observed throughout the development of wild- type plants and those expressing fasciation via scanning electron microscopy (SEM) and light microscopy. These observations revealed morphological and anatomical abnormalities of shoot apical meristem (SAM) development including an enlarged ring- shaped meristem, abnormal organs, and callus tissue. Comparing these observations with current literature for genetic and hormonal interactions in the SAM of plants, it is proposed that ring fasciation and callus formation in Clarkia tembloriensis occur as a result of abnormal environmental conditions via abnormal signaling of phytohormones and other developmental regulators which function in maintenance of meristem size and organ initiation.
A MORPHOLOGICAL AND ANATOMICAL INVESTIGATION OF SHOOT APICAL MERISTEMS EXPRESSING RING FASCIATION IN CLARKIA TEMBLORIENSIS
A Thesis
Submitted to the
Faculty of Miami University
in partial fulfillment of
the requirements for the degree of
Master of Science
by
Kilian TysonMayer
Miami University
Oxford, Ohio
2019
Advisor: Nancy L. Smith-Huerta
Reader: Alfredo J. Huerta
Reader: Carolyn H. Keiffer
©2019 Kilian TysonMayer
This Thesis titled
A MORPHOLOGICAL AND ANATOMICAL INVESTIGATION OF SHOOT APICAL MERISTEMS EXPRESSING RING FASCIATION IN CLARKIA TEMBLORIENSIS
by
Kilian TysonMayer
has been approved for publication by
The College of Arts and Science
and
Department of Biology
______Nancy L. Smith-Huerta
______Alfredo J. Huerta
______Carolyn H. Keiffer
Table of Contents
Introduction ...... 1 Materials and Methods ...... 7 Plant Culture ...... 7 Scanning Electron Microscopy, Light Microscopy, and Photography ...... 8 Observations of Growth in the Field ...... 8 Results ...... 9 Preliminary Observations of Abnormal Growth ...... 9 SEM Observations of SAM Development ...... 9 LM Observations of SAM Development ...... 11 Abnormal Floral Features ...... 12 Observations of Growth in the Field ...... 13 Discussion...... 13 Preliminary Observations of Abnormal Growth ...... 13 SEM Observations of SAM Development ...... 14 LM Observations of SAM Development ...... 18 Abnormal Floral Features ...... 20 Observations of Growth in the Field ...... 21 Conclusion ...... 22 Figures ...... 23 References ...... 35 Appendices ...... 44 Appendix 1 – Conceptual Diagram ...... 44 Appendix 2 – Apex Removal Experiment ...... 45
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List of Tables
Table 1. GPS Coordinates of Populations Visited...... 9 Supplemental Table 1. Results of Statistical Analyses for Apex Removal ...... 48 Supplemental Table 2. Summary of Apex Removal Experiment Data...... 48
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List of Figures
Figure 1. Digital Photographs of Morphological Abnormalities ...... 23 Figure 2. SEM Images of Early SAM Development ...... 24 Figure 3. SEM Images of Mid-Stage SAM Development ...... 25 Figure 4. SEM Images of Late SAM Development ...... 26 Figure 5. SEM Images of Apical Callus Development...... 27 Figure 6. LM Images of Early SAM Development ...... 28 Figure 7. High-Magnification LM Images of Mid-Stage SAM Development ...... 29 Figure 8. Low-Magnification LM Images of Mid-Stage SAM Development ...... 30 Figure 9. LM Images of Callus Tissue Development ...... 31 Figure 10. SEM Images of Abnormal Floral Features ...... 32 Figure 11. LM Image of Abnormal Floral Features ...... 33 Figure 12. LM Images of Ovule and Abnormal Stigma ...... 34
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Acknowledgements
Gratitude is extended to the Willard Sherman Turrell Herbarium at Miami University and its curator, Dr. Michael Vincent, for facilitating the grant which funded my field collections trip. I would also like to thank Ryan O’Dell from the Bureau of Land Management’s Hollister field office for granting access to their land and offering expert field identification and navigation to populations of Clarkia tembloriensis. Much appreciation is owed to Dr. Richard Edelmann and Matthew Duley of Miami University’s Center for Advanced Microscopy and Imaging for their consistent help to perfect sample preparation and imaging techniques. Without the CAMI facility, the SEM course, and their expert input, this project would not have been possible. I would also like to thank Chelsea Obrebski for informative discussions during image analyses and additional assistance in troubleshooting sample preparation and imaging issues. My lab work would likely have taken much longer if Chelsea had not begun to perfect her light microscopy sample preparation procedure a few months prior to mine. I am grateful to my parents, Karen Tyson and Timothy Mayer, for constant encouragement, intermittent financial support, and for setting me on the path to higher education. In addition, I would like to thank Dr. Robert Verb for initially sparking my interest in botany and Dr. Stephen Kolomyjec for consistently going out of his way to help others understand science by promoting a positive, open-discussion approach to educating the general public. Lastly, I would like to thank my advisor Dr. Nancy Smith-Huerta for guidance in all aspects of my project as well as for continued correspondence and academic support despite retiring before the completion of my project. Likewise, I am grateful to my committee members Dr. Alfredo Huerta and Dr. Carolyn Keiffer as well as my former committee member Dr. Daniel Gladish for guidance on experimental design and for helping me to better understand the nuances of experimental botany.
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Introduction
Variations in plant form have been the subject of scientific study for centuries to elucidate the details of plant organ development, and one such variation called fasciation has been artificially selected in agriculture for millennia (Moquin-Tandon 1841; Compton 1911; Johnson 1926; Bausor 1937; White 1948; Clark et al. 1993; Taguchi-Shiobara et al. 2001; Taiz 2013). Fasciation is an abnormal growth condition in plants which involves a change in shoot or root axis production from the normally round or polygonal form to one which is enlarged and irregular in shape (White 1948; Taguchi-Shiobara et al. 2001; Iliev and Kitin 2011; Reed et al. 2013). Fasciation can affect the morphology of any organ originating from the growing point, such as the stem, leaves, lateral roots, and flowers, and although this generates a wide variety of potential growth forms and degrees of expression, fasciation is most commonly recognized in shoots by large, flat stems and fan-like apices or inflorescences which can have undulating edges (White 1948; Majumdar 1955; Ansari and Daehler 2011; Reed et al. 2013; Mandel et al. 2014; Bommert and Whipple 2017). This type of expression is typically called band fasciation, but is sometimes referred to as cresting or cristation (Boke and Ross 1978; Iliev and Kitin 2011). Fasciation is always associated with an increase in biomass, whether the cause be an increased organ size or number, a trait that has targeted crops expressing fasciation for artificial selection in agriculture and led to the production of many familiar fruits and vegetables found at the grocery store today (White 1948; Taiz 2013). A few examples of these are the flowers and fruits of sweet corn, the fruits of tomato, the flowers and stems of broccoli, the terminal flower clusters of cauliflower, and many others. Aside from the obvious economic and agricultural benefits of increased crop yields, there is much to be gained from the scientific study of fasciation as described below. A thorough understanding of any variation in plant morphology may be crucial to advances in many areas of botany. In particular, it is important to understand how and why such variations develop because this fundamental knowledge illuminates a wider range of forces that affect plant growth in specific ways. Fasciation is an excellent phenomenon to use for the investigation of plant development in part because its occurrence is widespread throughout the flowering plants, documented in over 100 flowering plant families (White 1948; Tang and Skorupska 1997; Karakaya et al. 2002; Ansari and Daehler 2011; Iliev and Kitin 2011). In addition to the variety of plants expressing fasciation allowing utilization of many potential model organisms, the incredible diversity of growth forms resulting from that expression and the list of associated causes provide a labyrinth of research questions for potential investigation (White 1948; Clark et al. 1993; Tang and Skorupska 1997; Iliev and Kitin 2011; Reed et al. 2013). Fasciation may be expressed as a result of a single recessive gene, making investigation of the trait over multiple generations simpler in a lab setting. Examples of a single gene influencing expression of fasciation have been observed in a number of species including Arabidopsis thaliana (Leyser and Furner 1992; Mozgova et al. 2010), Glycine max (Tang and Skorupska 1997; Karakaya et al. 2002), Solanum lycopersicum (Barrero et al. 2006), Zea mays (Taguchi-Shiobara et al. 2001), and others (White 1948). Interestingly, fasciation was used as one of Mendel’s original heritable traits in the study of pea plants, playing a supporting role in the foundation of basic principles in the field of genetics (Taguchi-Shiobara et al. 2001; Iliev and Kitin 2011).
1
There is remarkable plasticity in form with the expression of fasciation, the major varieties including band, spontaneous, ring, multi-radiate, and bifurcating fasciation (White 1948; Majumdar 1955; Tang and Skorupska 1997; Singh et al. 2011; Mandel et al. 2014). Each type of fasciation appears to result from different patterns of floral or apical meristem development, all leading to the formation of more organ primordia and shorter internodes towards the apex. While there is much evidence to support the school of thought put forward by Moquin-Tandon (1841) that fasciation arises from changes in size and shape to a single growing point, it was suggested earlier by Linnaeus (1751) that fasciation arises due to extreme crowding and eventually fusion of several smaller growing points. The latter view has not gained nearly as much support since the early 20th century, though the idea has persisted in a few more-recent publications (White 1948; Kundu and Rao 1960; Sinjushin and Gostimsky 2006; Sinyushin 2010). Both shoot apical meristems (SAM) and root apical meristems (RAM) are known to express fasciation, but research focus has been largely on above-ground parts of the plant since recording observations and experimental procedures is easier and less invasive than in subterranean plant organs. As stated above, the abnormal growth of the SAM leading to fasciation may take on several different forms. Normal SAM development in dicotyledonous angiosperms maintains a radially symmetrical dome of undifferentiated cells (Beck 2010). In band fasciations, the SAM elongates along one axis resulting in a stem of approximately normal thickness in one dimension which becomes progressively wider in another (Tang and Skorupska 1997; Iliev and Kitin 2011). Also called linear fasciation, this is the most common type and is sometimes described as producing a ribbon-like stem because of its flattened appearance (White 1948). Spontaneous fasciation results in the SAM widening significantly in all directions rather than just one, resulting in thickened, club-like stems or enlarged inflorescences with a warped appearance (Tang and Skorupska 1997; Singh et al. 2011). Bifurcating fasciation is similar to banded expression in its early stages, but splits into two smaller SAMs rather than maintaining a single large one (White 1948). Multi-radiate fasciations are quite complex, resulting in the formation of many new apices from a single point in a “witch’s broom” effect, often following a band or spontaneous SAM fasciation (White 1948; Iliev and Kitin 2014). Ring fasciations are considered relatively rare in nature and develop much differently from those previously discussed. In ring fasciations, the center of the apical dome ceases or slows meristematic activity while the periphery continues to grow upward, resulting in a bowl or tube-like stem (Majumdar 1955; Taguchi-Shiobara 2001; Sinjushin and Gostimsky 2006; Singh et al. 2011). Meristem activity may be influenced by environmental factors such as atypical nutrient availability, excess water, physical damage, bacterial infection, and radiation, but these effects are facilitated by the complex interaction of genetic and hormonal signaling molecules responsible for regulating SAM maintentance (White 1948; Putnam and Miller 2007; Clark et al. 1993; Singh et al. 2011; Ansari and Daehler 2011; Man et al. 2011; Vernoux et al. 2011; Reed et al. 2013; Traas 2013; Cucinotta et al. 2014). High nutrient concentrations and water availability have been shown to increase the frequency of fasciation in many plants such as Clarkia, Lycopersicon, Oenothera, and Antirhhinum (White 1948; Reed et al. 2013; Smith-Huerta and Huerta 2015 Poster). Physical damage to apices has been documented to stimulate fasciation in a few plants (White 1948), but it has also been shown to inhibit expression in others (Ansari and Daehler 2011). Rhodococcus fascians is a bacterium that causes a wide range of abnormal growth forms, including fasciation, in at least 40 plant families (Putnam and Miller 2007). It is likely that the bacteria secrete some substance with an effect on the hormonal balances in cells 2 underlying the epidermis on which it resides. Exposure of seeds or young plants to various forms of radiation such as X-rays in Helianthus (Johnson 1926) and gamma rays in Gerbera jamesonii (Singh et al. 2011) also have the potential to stimulate fasciation. While the above environmental causes clearly have an effect on SAM activity, it is widely accepted that the underlying mechanism for fasciation is the abnormal action of hormonal and genetic factors regulating cell division and differentiation. Genes responsible for regulating floral and shoot meristem activity and size have been investigated in some model organisms such as Arabidopsis thaliana (Leyser and Furner 1992; Fletcher et al. 1999; Williams and Fletcher 2005; Iliev and Kitin 2011; Mandel et al. 2014; Bykova et al. 2016; Yamaguchi et al. 2017), tomato (Mertens and Burdick 1954; Barrero et al. 2006; Monforte et al. 2014; Van der Knaap et al. 2014; Xu et al. 2015), soybean (Tang and Skorupska 1997; Yamamoto et al. 2000; Karakaya et al. 2002; Mirzaei et al. 2017), and maize (Pautler et al. 2015; Yang et al. 2015; Je et al. 2016; Bommert and Whipple 2017; Je et al. 2018). While fasciation may be expressed due to a single recessive gene in many cases, there are multiple regulatory factors for which a mutation may elicit similar changes in SAM morphology (Clark et al. 1993; Shani et al. 2006; Iliev and Kitin 2011; Je et al. 2018). The most extensive genetic research has been done with Arabidopsis and has led to the characterization of several genes in a negative feedback loop regulating cell division and differentiation (Williams and Fletcher 2005). Arguably one of the most important to SAM fasciation is the WUSCHEL (WUS) homeodomain transcription factor responsible for maintaining a stem cell population near the organizing center (Carles and Fletcher 2003). This group of stem cells in the central zone of the corpus and lower tunica layers of an apical meristem is responsible for replenishing the surrounding cells which give rise to new primordia (peripheral zone) and stem growth (rib zone). Without proper WUS function, stem cells are mis-specified and terminate growth prematurely (Clark et al. 1993; Laux et al. 1996; Williams and Fletcher 2005). However, the stem cell population around the organizing center may continue to proliferate and result in overall SAM enlargement if WUS expression is not sufficiently suppressed (Williams et al. 2005). Several genes work to suppress WUS expression and therefore limit the size of the pluripotent cell population to the organizing center and overlying central zone. The most well-known of these genes include the ligand-receptor system of CLAVATA1 (CLV1) and CLV3 (Mandel et al. 2014). Stem cells above the organizing center express CLV3, secreting a signaling molecule that activates CLV1 expression in cells surrounding the organizing center, which suppress WUS expression (Williams and Fletcher 2005). Since increased WUS expression stimulates increased CLV3 expression, the reservoir of pluripotent cells maintains a relatively consistent size under normal conditions. Although the WUS-CLV feedback loop may be the primary regulatory system for SAM size in Arabidopsis, there are several other genetic factors which may cause similar irregularities, most commonly by direct interference with WUS, CLV1, or CLV3 (Leyser and Furner 1992; Clark et al. 1993; Williams et al. 2005; Mandel et al. 2014). Other genes affecting SAM size include ULTRAPETALA1 (ULT1), SPLAYED (SYD), HANABA TARANU (HAN), STIMPY/WOX9 (STIP), FASCIATA1 (FAS1), FAS2, phabulosa (PHB), phavoluta (PHV), CORONA (CNA), and ERECTA (ER) (Williams and Fletcher 2005; Mandel et al. 2014). The gene SYD is an SNF2-class chromatin-remodeling ATPase which upregulates the pluripotent stem cell pool in the organizing center by targeting WUS. Mutants for this gene have been found to prematurely terminate the SAM (Kwon et al. 2005; Williams and Fletcher 2005). The homeobox gene STIP is similar to WUS which also upregulates WUS expression in the organizing center but may be negatively regulated by CLV expression (Wu et 3 al. 2005). Mutants for STIP function grow flattened SAMs with differentiated tissue that lack WUS and CLV expression, but there appears to be a relatively complex interaction with CLV as a clv3 mutant phenotype may be completely suppressed by a loss of STIP function (Wu et al. 2005; Williams and Fletcher 2005). The gene ULT1 encodes a SAND-domain transcriptional regulator which restricts size in both floral and shoot meristems, at least partly by controlling the CLV1 expression domain to restrict that of WUS (Fletcher 2001; Carles et al. 2005). Early in SAM development, the GATA-3-like transcription factor HAN is responsible for restricting the WUS expression domain before CLV expression is initiated (Williams and Fletcher 2005). The genes FAS1 and FAS2 encode two subunits of CHROMATIN ASSEMBLY FACTOR-1 (CAF-1) responsible for maintenance of cellular and functional organization in both shoot and root meristems, and a mutation in either gene results in ectopic WUS expression and its defective maintenance (Kaya et al. 2001; Williams and Fletcher 2005). The genes PHB, PHV, and CNA are members of the Class III homeodomain-leucine zipper family that serve a variety of complex regulatory functions in meristematic activity, but also appear to suppress WUS transcription as triple mutants show SAM growth similar to those with reduced CLV function (Leyser and Furner 1992; Prigge et al. 2005; Williams and Fletcher 2005). Another receptor kinase that acts similar to but independently from CLV to regulate WUS expression in the SAM is ER. Reduced activity of ER may lead to ectopic carpel development on floral meristems by increased expression of AGAMOUS (AG) (Clark et al. 1993; Mandel et al. 2014). It has also been shown that overexpression of microRNA miR166g causes increased WUS expression and that other mutations in genes such as LEAFY (LFY) and APETALA1 (AP1) can enhance fasciated phenotypes that may be caused by reduced CLV expression (Clark et al. 1993; Williams et al. 2005). The complex network of genetic factors regulating and affecting meristem activity clearly has the capability to stimulate fasciation by a variety of pathways. The role that plant hormones play in the development of the SAM is a critical area of consideration relevant to an investigation of fasciation. Of the known phytohormones, auxin is perhaps the most strongly-associated with regulation of SAM activity, particularly primordium initiation and the regular spacing of lateral organs, known as phyllotaxis (Reinhardt et al. 2003; Shani et al. 2006; Traas 2013). There has been support for several models of phyllotaxis regulation including mechanisms such as biophysical forces and inhibitors of organogenesis restricting primordium formation (Snow and Snow 1962; Mitchison 1977; Green 1992; Selker et al. 1992; Green 1996; Green 1999). Many of the biophysical models are supported heavily by theoretical mathematical calculations, and recent years have led to much more experimental support for mechanisms based on auxin signaling and transport (Benkova et al. 2003; Reinhardt et al. 2003; Heisler et al. 2005; Shani et al. 2006; Barbier de Reuille et al. 2006; Vernoux et al. 2011; Traas 2013). The most abundant naturally-occurring form of auxin in plants is indole-3- acetic acid (IAA), which is not able to diffuse freely through cell membranes in its ionized form and so is transported by specialized influx and efflux carriers known as AUX/LAX and PIN proteins, respectively (Grunewald and Friml 2010). While influx carriers are distributed relatively uniformly in the plasma membrane, efflux carriers are localized to one side of the cell which facilitates directional (polar) transport (Traas 2013). Polar transport of auxin is crucial for the regulation of phyllotaxis, and this has been shown by a variety of defects in patterning brought about by interference with auxin transport or response in Arabidopsis (Reinhardt et al. 2003). Under normal conditions, the pattern of transport in the SAM leads to a non-uniform distribution of auxin in high concentrations (maxima) and low concentrations (minima) which appear to determine the position of incipient primordia such that maxima indicate new growth
4 while minima predict spaces between them (Barbier de Reuille et al. 2006; Shani et al. 2006; Grunewald and Friml 2010; Traas 2013). Growing primordia function as local sinks for auxin in the SAM periphery, depleting the available auxin in a certain minimal area surrounding each primordium and preventing additional primordium formation except at a certain minimal distance (Reinhardt et al. 2003). This phenomenon explains many of the previous models for regulation of phyllotaxis such as minimum-energy buckling, inhibitors of organogenesis, and first-available space (Snow and Snow 1962; Mitchison 1977; Selker et al. 1992; Green 1996). Given the control of primordium initiation by auxin at the SAM and the morphological similarities between organs initiated with irregular auxin transport or response and those initiated by a fasciated SAM, it is likely that auxin plays a role in fasciation. Cytokinin is a plant hormone generally associated with stimulating cell division in plants but plays a particularly important role in SAM maintenance (Ferreira and Kieber 2005; Shani et al. 2006). Arabidopsis HISTIDINE KINASE 2 (AHK2), AHK3, and AHK4 are three genes known to encode cytokinin receptors in the SAM. Triple mutants for these genes have severely impaired growth and SAM size associated with reduced meristematic activity while single and double mutants have wild-type or slightly impaired growth. This suggests partially redundant roles in cytokinin sensitivity for these genes, also suggesting that cytokinin prevents differentiation of meristematic cells in the SAM and young primordia (Nishimura et al. 2004). Reduction in cytokinin levels by increasing the cytokinin-degrading enzyme cytokinin oxidase results in reduced meristem size and leaf initiation rate, occasionally to the point of meristem abortion, further supporting a critical regulatory role for cytokinin in SAM activity (Werner et al. 2003). Cytokinin activity and distribution in the SAM are also affected by patterns of WUS and KNOX1 transcription factors as well as Arabidopsis type A response regulators (ARR), resulting in localized gradients of cytokinin content and response with a range of effects (Shani et al. 2006). Cytokinin is known to interact with auxin to regulate SAM processes in a variety of ways (Schaller et al. 2015). Although many pathways are yet unclear, the two hormones have been shown to maintain a balance in WUS expression at the organizing center. While cytokinin upregulates ARR expression, auxin provides a negative regulation which increases sensitivity to cytokinin and encourages proliferation of meristematic cells (Zhao et al. 2010). In addition, expression of WUS negatively regulates ARRs for a similar effect while cytokinin is required in relatively high concentrations for proper WUS expression in the organizing center (Schaller et al. 2015). Cytokinin has been shown to directly affect the polar transport of auxin by enhancing transporter depletion at specific polar domains, effectively rearranging the direction of auxin flow and leading to lateral organ initiation (Marhavy et al. 2014). On the other hand, at least one inhibitor of cytokinin signaling (AHP6) is regulated downstream of auxin and is regarded as a critical component to maintaining the complex auxin and cytokinin fields responsible for initiation of organ primordia and regulating phyllotaxis (Besnard et al. 2014). Auxin distribution and response has also been shown to affect cytokinin biosynthesis and therefore organogenesis (Cheng et al. 2013). Given the complexity of hormonal interactions in the SAM which affect cell identity and division, it is certain that cytokinin signaling has the capability to suppress or enhance expression of fasciation in plants. This is well-supported by experiments demonstrating fasciated phenotypes associated with high cytokinin both naturally occurring and exogenously applied (Varga et al. 1988; Nilsson et al. 1996; Papafotiou et al. 2001; McCartan and Van Staden 2003; Kitin et al. 2005; Mitras et al. 2009; Iliev and Kitin 2011). Gibberellins are a group of plant hormones that function largely in opposition to cytokinin in the SAM to further increase the complexity of hormonal gradients and boundaries 5
(Shani et al. 2006). The most common form of the many gibberelins in plants is known as gibberellic acid (GA3). Increased naturally occurring concentrations and exogenous application have been shown to stimulate fasciation in cotton similar to the effects of cytokinin on many species, suggesting that gibberellins interact with a complex network of hormonal and genetic signals (Nadjimov et al. 1999; Iliev and Kitin 2011). Although much has yet to be elucidated about the role of gibberellins in SAMs, evidence suggests one primary function may be determination of the boundary between the apical dome and the incipient primordium (Shani et al. 2006). Although they are not fully understood in the context of SAM development, other phytohormones such as ethylene and brassinosteroids have been shown to play important roles in many aspects of plant growth and are likely to interact with and influence signaling pathways regulating the SAM (Clouse et al. 1996; Clouse and Sasse 1998; Shani et al. 2006; Cheng et al. 2013). Like gibberellins, brassinosteroids have been implicated as an important regulator in organ boundary formation (Gendron et al. 2012; Cucinotta et al. 2014). Callus is a term used to collectively refer to various disorganized masses of undifferentiated cells in plants (Ikeuchi et al. 2013). Callus development in plants is similar to fasciation in that a variety of environmental stimuli can disrupt key molecular regulation systems which maintain cell differentiation, leading to excessive proliferation of callus tissue. In fasciation this is limited to existing meristems which expand their undifferentiated region in any of several morphological patterns described above, but callus tissue may develop from most living cells in the plant body (Ikeuchi et al. 2013). Like fasciation, there are multiple types of callus that have been identified as distinct based on tissue morphology and organ regeneration capabilities. These include calli with and without organ regeneration, calli with compact and loose (friable) tissue morphology, calli grown on callus-inducing-medium (CIM) and those induced by wounding, and calli that give rise to shoots (shooty), reproductive organs (embryonic), and roots (rooty) (Ikeuchi et al. 2013). Due to the similar mechanisms by which the development of fasciation and callus take place (i.e. disruption of hormonal and genetic regulatory framework), there are overlapping biotic and abiotic stimuli for the growth conditions such as physical wounding and bacteria which insert genes that encode biosynthetic enzymes for auxin and cytokinin into host plant cells. Both conditions are extremely sensitive to auxin and cytokinin concentration and share one critical gene for the regulation of undifferentiated tissue proliferation in Arabidopsis: WUSCHEL (Ikeuchi et al. 2013). Ectopic expression of the SAM regulatory genes SHOOTMERISTEMLESS and CLAVATA3 has also been documented in callus tissue from mutant plants deficient in the deposition of cell wall structural material (Krupkova and Schmulling 2009). Deficiencies in cell wall structural deposition also affect callus structure, resulting in more disorganized morphology and weak intercellular attachment (Iwai et al. 2002). Mechanical damage has been cited as a cause for both fasciation and callus formation, but research to date has only elucidated the molecular pathway mediating callus formation, a pathway which occurs much more consistently and is dependent on the transcription factor WOUND INDUCED DEDIFFERENTIATION1 (WIND1) and its homologs WIND2-4 (Ikeuchi et al. 2013). Given that expression of SAM fasciation typically entails abnormal signaling of auxin, cytokinin, and WUS (Clark et al. 1993; Iliev and Kitin 2011), it is likely that fasciation is a sufficient disruption to molecular signaling that it may be involved in inducing callus development at the apex. Clearly, much has been learned over the years about the nature of fasciation in plants. The recent laboratory studies of model organisms such as Arabidopsis, soybean, tomato, and corn described above have helped to elucidate the environmental, genetic, and hormonal factors 6 regulating SAM activity (Leyser and Furner 1992; Clark et al. 1993; Tang and Skorupska 1997; Ansari and Daehler 2011; Mandel et al. 2014; Xu et al. 2015; Je et al. 2016; Je et al. 2018). In addition to these laboratory studies of fasciation, it is interesting and important to consider the nature of fasciation as it relates to the evolution of plant form in wild and agricultural systems. As mentioned earlier, fasciation has played an important role in morphological changes in agricultural plants (White 1948; Taguchi-Shiobara et al. 2001; Taiz 2013). The occurrence and nature of fasciation in natural plant populations has also been the subject of study (White 1948; Ansari and Daehler 2011; Iliev and Kitin 2011). One such instance of a wild plant expressing fasciation when grown in laboratory conditions different from its natural environment is Clarkia tembloriensis V. (Onagraceae) (Smith-Huerta and Huerta 2015; Smith-Huerta and Huerta 2015 Poster). This wildflower is endemic to only 10 counties in the semi-arid western foothills of the San Joaquin Valley in California (Calflora 2018) and has been used to investigate population genetics and floral development, revealing variation in both outcrossing rates and flower morphology between natural populations (Vasek and Harding 1976; Holtsford and Ellstrand 1989; Holtsford and Ellstrand 1990; Holtsford and Ellstrand 1992; Smith-Huerta 1992; Holtsford 1996; Sherry and Lord 1996; Smith-Huerta 1996; Kerwin and Smith-Huerta 2000; Sherry and Lord 2000). Additionally, observation of plants grown from seeds of one population (Crocker Canyon) in growth chamber conditions has identified a phenotype which appears to express stem fasciation (Smith-Huerta and Huerta 2015; Smith-Huerta and Huerta 2015 Poster). Due to the water-limited conditions of its natural habitat which undergoes relatively frequent and severe droughts, it was hypothesized that the increased water availability provided by growth chamber conditions stimulated fasciation in C. tembloriensis by altering the balance of hormonal signals at the apex. This sensitivity to an easily manipulated potential environmental cause of fasciation in conjunction with a genetic predisposition for its expression and an annual life cycle make C. tembloriensis an excellent model organism to study SAM fasciation. There were three goals for this project. The first was to characterize the developmental morphology of fasciation in C. tembloriensis using Scanning Electron Microscopy (SEM) to visualize the SAM surface with a three-dimensional perspective. The second was to use Light Microscopy (LM) to gain an understanding of underlying meristem anatomy associated with the change characterized by SEM. The third goal was to determine if fasciation is expressed in natural populations following a growth season with heavy rainfall.
Methods
Plant Culture Seeds for three genotypes of experimental Clarkia tembloriensis plants were collected from the California populations of Cantua Creek (field-collected 2013) and Crocker Canyon (chamber-grown 2014). The Cantua Creek population (GPS coordinates: 36.4137619, - 120.47450639) described by Holtsford and Ellstrand (1989) as CC1 was chosen as a control because it consistently displays wild-type morphology with respect to phenotypes of both SAM fasciation and the homeotic mutant crinkled petal (cp) gene (Smith-Huerta 1992; Leong et al. 2001; Smith-Huerta and Huerta 2015). The Crocker Canyon population (GPS coordinates: 35.215617, -119.690917) is polymorphic for expression of the cp phenotype, and was chosen as an experimental group because progeny of these plants have been observed to express fasciation 7 in well-watered growth chamber conditions (Smith-Huerta and Huerta 2015; Smith-Huerta and Huerta 2015 Poster). Seeds from the Crocker Canyon population expressing the cp phenotype (CCP) were distinguished from those with normal flower petals (CWT) in this study because the cp gene has been demonstrated to influence the expression of fasciation in C. tembloriensis (Smith-Huerta and Huerta 2015; Smith-Huerta and Huerta 2015 Poster). All experimental plants were grown from seed to maturity with fluorescent lights in climate-controlled growth chambers (20º C on a 12-hour light/dark cycle) at Miami University in Oxford, Ohio. For germination, seeds were sprinkled onto the surface of Thermo-O-Rock vermiculite contained in 3-inch pots. The pots containing the vermiculite and seeds were watered gently and allowed to sit in trays containing approximately 1 inch of water, then allowed to grow until the first true leaves began to expand (2-4 weeks). Seedlings were then transplanted into 6-inch pots with Fafard 3B© Soil Mix (Sun Gro Horticulture 770 Silver Street Agawam, MA 01001). The amount of water provided and the number of plants per pot varied by treatment and experiment (see below for details). Scanning Electron Microscopy, Light Microscopy, and Photography Plants for both scanning electron microscopy (SEM) and light microscopy (LM) included 60 chamber-grown descendants of the CC1 population and 60 chamber-grown descendants of the Crocker Canyon population (CWT). Plants chosen for photography included 20 plants of the 3 genotypes (3 CC1, 7 CWT, 10 CCP) from preliminary observation groups prior to apex preservation. Plants used in photography were transplanted in a variety of groupings (i.e. 1-10 plants per pot) and watered 1-2 times per week for approximately 3 months before photographs were taken. Seedlings for SEM and LM were transplanted to soil (2 plants per pot) and spatially randomized into three groups with a random number generator. All plants for SEM and LM were well-watered and grown over the course of approximately 2 months following transplanting. Twelve shoot apices were harvested for SEM and LM every three to four days (6 CC1 and 6 CWT). All experimental plant material for SEM and LM was fixed in FAA (Johansen 1940) made using 70% ethanol and processed for sectioning and microscopy by serial dehydration with ethanol. The samples used for SEM were then critical-point-dried and sputter-coated with ~20 nm of gold particles for viewing using a JEOL-840A scanning electron microscope in the Center for Advanced Microscopy and Imaging (CAMI) at Miami University. Samples for LM were embedded in Spurr’s resin (Spurr 1969) over several days, cut into 1.5 µm sections with a diamond knife and microtome, then stained with a Toluidine blue solution (Smith and Hake 2003) and sealed onto glass slides for viewing in an Olympus AX-70 light microscope in the CAMI at Miami University. Scanning electron microscopy and light microscopy were used to visualize the SAM surface and internal anatomy at various stages during development of fasciation in CWT and normal apical development in CC1. Photomicrographs of selected specimens were used to construct figures representative of each observed stage in both normal and fasciated development, then compared based on morphology and anatomy. In a few cases, observed morphological abnormalities associated with later development of fasciation in CWT were represented in figures by CCP plants outside of the main experimental group due to superior image quality. This was only done with CCP plants that were grown under the same conditions, were older than 60 days after transplanting (DAT), and which obviously displayed particular features observed in CWT apices. These substitutions were considered reasonable because the observed developmental pattern of CWT and CCP SAMs expressing fasciation was generally very similar with the exception that CCP SAMs appeared to be more responsive to environmental stimulation, either temporally or in degree of enlargement (personal 8 observations). Although Smith-Huerta and Huerta (2015 Poster) described some statistically significant differences in fruit production and height between CWT and CCP plants given a well- watered treatment, these differences are consistent with the idea that CCP plants have comparatively increased phenotypic expression of fasciation (i.e. larger meristems devote more resources to stem thickness, resulting in shorter shoots with proportionally more flowers in the apical cluster). Digital pictures of particularly striking morphological abnormalities were taken to document variation in form during preliminary observations. Observations of Growth in the Field A field collection trip to the native habitat of C. tembloriensis was undertaken to document the expression of fasciation under natural conditions following the heavy rain of the 2016 El Niño. Specimens from each of 9 populations (Table 1) were collected and pressed for the Willard Sherman Turrell Herbarium in July of 2016 and very general environmental observations (i.e. temperature, general soil composition, and water availability) were made to assess any additional environmental stresses that might affect plant growth. Collected specimens largely consisted of dried shoot remains due to the spring flowering period ending approximately 2 months beforehand. Specimens were visually examined for signs of fasciation upon collection.
Table 1. Clarkia tembloriensis Populations Visited in July 2016 Population Name GPS Coordinates County Cantua Creek (CC1) 36.4137619, -120.47450639 Fresno Monocline Ridge (East) 36.53690962, -120.5311127 Fresno Monocline Ridge 36.53345712, -120.5639004 Fresno Tumey Hills 36.61580181, -120.65558009 Fresno Panoche 36.71562986, -120.82641483 Fresno Idria (Pimental Creek) 36.53737652, -120.83498144 San Benito Idria (Griswold Hills) 36.5283686, -120.81670734 San Benito McKittrick 35.34447413, -119.84081089 San Luis Obispo Crocker Canyon 35.215617, -119.690917 San Luis Obispo Elkhorn 35.011082, -119.462882 Kern *GPS coordinates given in decimal degrees
Results
Preliminary Observations of Abnormal Growth When Clarkia tembloriensis plants descended from the Cantua Creek (CC1) and Crocker Canyon populations (CWT and CCP) were grown under growing conditions different from those found in nature (in growth chambers with abundant water and nutrients in addition to a growing period of 4-6 months rather than the typical 3 months), numerous developmental abnormalities were observed (fig. 1). With respect to fasciation, shoot apices occasionally exhibited flat stems associated with band fasciation (fig. 1A) and commonly produced one to several new shoots from the point of SAM fasciation on the main axis (fig. 1B). New shoots from fasciated apices produced organs ranging from those with normal morphology to organs with abnormal
9 curvature, stunted development, or reduced filamentous structure (fig. 1B). The new shoots were observed very late in development and could be relatively straight, normal axes or could become flat and curved, often appearing to loop back towards the point of origin (fig. 1C). When these shoots became thickened but remained straight, they often bifurcated with further growth (fig. 1A). Most fasciated apices appeared to have a club-like apex with many organs clustered at the top of an increasingly thickened stem. Often, this apex would produce leaves with abnormal curvature and spongey, white, callus-like growths associated with the inner edge or primary vein in a marginal location (fig. 1D). SEM Observations of SAM Development All plants (CC1 and CWT) began with normal SAM morphology and an apical dome approximately 100 µm in diameter (fig. 2A-B). In plants expressing fasciation, normal development was observed to last up to 35 DAT. At around 30-40 DAT, CWT plants expressing fasciation began to display a widened SAM (fig. 2D) and shorter internodes resulting in more leaf and floral primordia close to the apex when compared with normal CC1 plants (fig. 2C-D). Approximately 35-45 DAT, the apical dome of plants expressing fasciation appeared to become relatively flat and developed an undulating surface (fig. 2F). Following SAM enlargement in CWT plants expressing fasciation (40-50 DAT), surface cells in the center of the apical dome produced trichomes (fig. 3B). These cells are part of the L1 tunica layer and were not observed to produce trichomes during normal development of Cantua SAMs (fig. 3A, 3C). The central zone of the SAM expressing fasciation appeared to cease growth shortly after tunica differentiation while the peripheral zone remained meristematic, causing the periphery to form a ring shape at approximately 45-55 DAT (fig. 3D). At this point, the difference in SAM size between normal plants and those expressing fasciation became even greater and more trichomes developed in the middle and outer periphery of the SAM expressing fasciation (fig. 3C-D). Further development of the ring-shaped SAM was quickly accompanied by an increased number of leaf and floral primordia in the apical cluster at approximately 45-55 DAT, most likely due to a combination of altered phyllotaxy and reduced longitudinal growth of the stem. The ring-shaped SAM of CWT plants expressing fasciation was also dramatically larger than the SAM of CC1 plants with normal morphology (fig. 4A-B). While earlier ring morphology displayed a clearly raised periphery (fig. 3D), that periphery appeared to become continuous with the increasingly clustered leaf and floral primordia (fig. 4B). Many of these primordia developed with abnormal morphology, such as leaf primordia with margins that curved inward or disproportionally to one side, although these organs were removed during preparation to expose the apical ring or cavity (fig. 4B). Continued development of fasciated CWT apices resulted in the formation of friable callus tissue on the enlarged SAM following 50 DAT, which spread into the central cavity and onto the edges of surrounding organs (fig. 4D, 4F). Abnormal floral organs developed at the apex at a similar time and typically consisted of single, unfused carpel ovaries with an outer ovary wall that never fully enclosed ovules, instead demonstrating an irregular morphology reminiscent of an abnormal leaf or sepal with both margins inwardly curved at the base and at least partially fused with the apical callus tissue while the tip grew laterally away from it favoring one margin to produce a twisting morphology (fig. 4D, 4F). These and other abnormal floral organs are described in more detail in a later section. After the appearance and proliferation of friable callus tissue on the SAM surface of fasciated CWT plants (50+ DAT), callus tissue continued to divide and expand the apex diameter to many times the size of normal
10
CC1 apices (fig. 4E-F). During this period of apical callus tissue proliferation, the ring structure disappeared and the initiation of new organs became much less frequent as evidenced by an abundance of larger organs (and lack of new primordia) at the periphery (figs. 4F, 5A). New organs that were initiated during or immediately prior to apical callus proliferation typically developed with abnormally warped, fused, stunted, or pin-like morphology (fig. 4F), likely due to physical crowding of developing organs and disrupted hormonal signaling at the apex associated with callus development. As callus tissue proliferated inside the central cavity and throughout the SAM surface, apex morphology took on a relatively flat or slightly convex appearance (figs. 4F, 5B) rather than maintaining a concave bowl shape (fig. 5C). Apices that did maintain a concave morphology during more advanced callus proliferation often had a visible central cavity and callus tissue that was at least partially obstructed by growth of peripheral organs (fig. 5A), requiring more aggressive dissection in order to gain a view the interior surface (fig. 5C). Apical callus tissue consisted of loosely packed cells of normal size (approximately 10- 20 µm diameter) to significantly enlarged cell size (approximately 20-40 µm diameter) with no apparent organization at the surface (fig. 5B-C). Callus tissue that developed on surrounding organs was typically composed of cells with a more moderate size that appeared to facilitate peripheral organ fusion to the apical callus tissue or adjacent organs (fig. 4D, 4F). LM Observations of SAM Development Both CC1 and CWT plants began with normal SAM anatomy and an apical dome approximately 100 µm in diameter (fig. 6A-B). In CWT plants expressing fasciation, this was estimated to last up to 35 DAT. At approximately 30-40 DAT, plants expressing fasciation began to display an increased apical dome diameter compared to normal SAMs (fig. 6C-D). At approximately 35-45 DAT, the apical dome of plants expressing fasciation became relatively flat (fig. 6F). Plants expressing fasciation showed an increased diameter for the tunica layers, corpus, organizing center, and rib meristem compared to normal plants (fig. 6E-F) and reduced meristematic activity in the central cells of the L1 tunica layer (fig. 6F). Upper tunica layers appeared to remain 1 cell layer thick during early development in both CC1 and CWT, with easily distinguishable L1 and L2 layers above an L3 layer that was continuous with the underlying corpus and organizing center (fig. 6A-F). Meristematic activity in the middle of the organizing center in enlarged SAMs appeared to be slightly reduced while its margins and more peripheral corpus tissue maintained darkly staining cytoplasm (fig. 6F). Due to the high affinity of toluidine blue for acidic molecules, darkly staining cytoplasm likely indicates higher ribosome content or cytoskeletal components used during periods of increased metabolic activity. Plants developing normally maintained a dome-like SAM approximately 100 µm in diameter with anatomy generally adhering to the tunica-corpus model (fig. 7A, 7C). After early SAM enlargement in CWT plants expressing fasciation, cells in the L1 tunica layer began to produce trichomes at approximately 40-50 DAT (fig. 7B, 7D). At a similar time shortly after early SAM enlargement, the highly meristematic corpus region appeared to drastically increase its rate of proliferation to become many cell layers thick while still increasing in diameter, leading to an even greater difference in size between normal CC1 apices (fig. 8A, 8C)and CWT apices expressing fasciation (fig. 8B, 8D)). The large cell size and darkly-staining cytoplasm occupying a high proportion of intracellular space of most cells in the rib meristem in addition to the cluster of organs that were visible at the apex show that the enlarged corpus region did not contribute significantly to longitudinal stem growth (fig. 8B). Instead, meristematic proliferation appeared to be devoted largely to increasing SAM size by enlarging the pool of undifferentiated
11 meristematic cells known as the organizing center, increasing the number of cells in the upper corpus and lower tunica layers, and facilitating initiation and growth of organs on the clustered periphery (fig. 8B). At approximately 45-55 DAT, some callus tissue began to develop on the surface of SAMs that were expressing fasciation (fig. 7B, 7D). This tissue appeared to be part of the L1 tunica layer where the cells initially developed anatomical abnormalities including irregular shape, darkly staining cytoplasm, increased deposition of integument or cell wall material, and decreased intercellular organization resulting in a highly textured surface (fig. 7B, 7D). As enlargement of the corpus continued at approximately 45-55 DAT, apex diameter in CWT plants expressing fasciation increased continually (fig. 8B, 8D) while normal CC1 apices maintained a consistent apical dome diameter of approximately 100 µm (fig. 8A, 8C). The stain darkness of cells in the center of the enlarged corpus was reduced, indicating a higher rate of division for cells in the outer corpus and tunica layers (fig. 8D). The disproportionally peripheral proliferation of the enlarged corpus appeared to facilitate formation of the ring-shaped SAM surface by continually increasing the diameter of the apex, pushing the base of clustered organs further away from the center, though sufficiently large organs continued to elongate inward and obstruct the view of the central SAM surface. At the same time, the darkly staining cytoplasm of the upper corpus and lower tunica layers below the ring-like SAM surface indicated a higher rate of cell division in this region, which was responsible for rapid enlargement of this feature (fig. 8D). Cells in the center of the L1 and L2 layers showed very little cytoplasmic activity and were likely to be differentiated or in a non-dividing state, effectively stunting growth of the central SAM at the surface relative to the peripheral zone which continued meristematic proliferation (fig. 8D). The enlarged organizing center of CWT plants expressing fasciation appeared to remain visible immediately above the rib meristem (fig. 8B, 8D, which was also considerably wider than a normal CC1 rib meristem (fig. 8A, 8C). The enlarged organizing center demonstrated inconsistent patterns of cell division at this time, making it appear skewed or difficult to identify (fig. 8B, 8D). The topmost cell layers in the rib meristem appeared to be fully differentiated and enlarging with primarily vacuolar space, further evidenced by raphide production unusually close to the organizing center (fig. 8B), and further supporting the observation that the actively dividing cells contributed very little to longitudinal stem growth. It is also within this timeframe that organs in the apical cluster began to show abnormal shape and orientation due to crowding (fig. 8B). Further development of the ring-shaped SAM was often characterized by drastic size increases that were not conducive to proper infiltration with Spurr’s resin. Development of callus tissue on apices expressing fasciation in late stages appeared to take the form of extremely large, loosely packed cells with little to no cytoplasm visible, indicating largely vacuolar space (fig. 9A). The callus cells at the surface had a complex wall shape and it is possible this was due to increased sensitivity to osmolarity changes during sample preparation. The interior tissue layers appeared to transition seamlessly to stem tissue of normal cell size and shape, also differentiated, and with only a small portion of intracellular space containing cytoplasm (fig. 9A). Callus tissue on organs immediately surrounding the apical surface appeared to begin as small, loosely-packed cells filled with dense cytoplasm (i.e. fusion tissue) which then divided several times, maturing to an increased size and proportion of unoccupied intracellular space (fig. 9B). Abnormal Floral Features
12
Abnormal floral features initiated at the apex during later stages of expressing fasciation included flowers that display a range of abnormalities. In some cases (fig. 4D, 4F) these consisted of single, unfused carpels that surrounded an enlarged ring-like SAM or apical callus tissue. These single, unfused carpels had an outer ovary wall that never fully enclosed the ovules, instead demonstrating an irregular morphology with both margins inwardly curved at the base and at least partially fused to the callus tissue at the periphery of the apex while the tip grew laterally away favoring one margin to produce a twisting morphology (fig. 4D, 4F). Abnormal carpels initiated ovules on the interior side most closely associated with the apex (figs. 4F) (i.e. ovary wall margin with more fusion to apex). Another type of abnormal flower observed at the apex of CWT plants expressing fasciation consisted of unfused carpels and an abnormal style and stigma. The style and stigma arose from a portion of the interior periphery of an unfused carpel (fig. 10A-B, 11). Carpels that developed around the abnormal stigma and style had the same partially unfused morphology as the single, unfused carpels often observed to develop on late ring-like SAMs and near the expanding masses of apical callus tissue described above (fig. 4D, 4F). The style and stigma often retained callus tissue on the surface during initial growth which facilitated fusion to the interior edges of carpel ovaries (fig. 10A-B). A subset of this abnormal flower type was one that consisted of four carpels (the normal number for Clarkia tembloriensis) fused completely in some areas and unfused in others (fig. 11). In some instances, ovules appeared to be initiated from abnormal style tissue and at the fusion boundary between the style and partially unfused carpel walls (fig. 10A). In other instances, ovules were initiated on the central column of an abnormal ovary (fig. 11). Many of these ovules appeared to be in early stages of development with normal anatomy (fig. 12A), though it is unknown whether they could have eventually reached maturity. During style elongation, the surface tissue of the abnormal stigma was consistent with early callus formation and fusion tissue found on organs most proximal to apical callus (fig. 12B). Observations of Growth in the Field Field collections (Table 1) did not result in the observation of fasciation under natural conditions. However, plants remaining at the time of collection were mostly dried stalks, and herbivores appeared to have decimated two of the southern populations (including Crocker Canyon and McKittrick), leaving only a few small stems or decaying material from previous growing seasons. The Monocline Ridge populations appeared to remain in flowering stages much later than others, indicated by the presence of several live plants with intact flowers at the time of collection. In sites where a higher number of plants remained (including Cantua Creek and Tumey Hills), dry stalks were occasionally observed with a much taller and thicker stem than would be expected. However, none of these large plants had an apex morphology indicative of fasciation, and none of the populations where they were found are known to express fasciation. No major differences in temperature, general soil composition, or water availability were observed between the populations.
Discussion
Preliminary Observations of Abnormal Growth
13
The variety of abnormal organ morphologies in C. tembloriensis SAMs expressing fasciation under a prolonged lifecycle (4-6 months rather than the normal 3 months) in growth chamber conditions (figs. 1A-D) is consistent with irregular phytohormone signaling (Putnam and Miller 2007; Iliev and Kitin 2011; Gendron et al. 2012; Choob and Sinyushin 2012; Mandel et al. 2014). Phytohormone concentration is known to affect cell identity signaling at the apex such that the pluripotent pool of cells in the center of the SAM changes in size (i.e. fasciation) (Nilsson et al. 1997; Ferreira and Kieber 2005; Shani et al. 2006; Shi et al. 2018). It is also known to induce callus formation (Cheng et al. 2013; Ikeuchi et al. 2013; Ng et al. 2016) and affect organ morphology during development (Flemming 2005; Iliev and Kitin 2011; Mandel et al. 2014). Auxin and cytokinin have been shown to be crucial for proper meristem maintenance and early organ development (Shani et al. 2006; Iliev and Kitin 2011; Cheng et al. 2013; Besnard et al. 2014; Mandel et al. 2014; Schaller et al. 2015; Shi et al. 2018). Atypical brassinosteroid levels are associated with organ boundary formation deviating from normal development (Gendron et al. 2012). In the C. tembloriensis plants of this study, the frequently abnormal stem morphology of new shoots that grew from fasciated apices (figs. 1A-C) suggests that irregular phytohormone signaling at the apex was severe and widespread in late stages of expression. SEM Observations of SAM Development Normal SAM development for dicotyledonous angiosperms with spiral phyllotaxy refers to the centralized dome of undifferentiated cells that varies in size and shape among different species (Beck 2010). Many herbaceous angiosperms share a similar apical dome morphology during primary axis growth including Arabidopsis thaliana, the most commonly-used model organism in plant science (Tang and Skorupska 1997; Sinjushin and Gostimsky 2006; Zhao et al. 2010; Mandel et al. 2014). This normal apical dome morphology appears as a relatively symmetrical, rounded to flat group of cells at the uppermost center of the growing primary axis between 50 µm and 150 µm in diameter (Clark et al. 1993; Vernoux et al. 2000; Kwon et al. 2005; Williams et al. 2005). Clarkia tembloriensis appears to adhere to this apical dome morphology throughout normal development with a diameter of approximately 100 µm. In CWT plants expressing fasciation this was estimated to last until 30-40 DAT (fig. 2A-D), much later than observations of genetic mutant plants known to express fasciation (Clark et al. 1993; Tang and Skorupska 1997). Comparatively, exogenous application of phytohormones or synthetic compounds affecting hormone signaling at the apex affects SAM size quickly following the application and has even been known to induce ring fasciation (Majumdar 1955; Iliev and Kitin 2011; Bykova et al. 2016). Given the environmental stimulus for fasciation in this experiment (i.e. differences in available water and nutrients in addition to light exposure deviating from that of a natural environment) and the known involvement of phytohormones in molecular pathways for environmental signaling in plants (Man et al. 2011), the timing of initial SAM widening under consistent conditions supports the idea that phytohormones are a key factor for expressing fasciation in C. tembloriensis. The lack of fasciation by plants of the CC1 population under the same growing conditions further suggests that the Crocker Canyon population carries a genetic predisposition to disruption of meristem maintenance. Given the scope of this study, it is not clear whether the genetic difference is part of a sensitivity threshold for phytohormone concentration, a loss of function in one or more partially redundant genes involved in the WUS- CLV regulatory feedback loop, or a pathway independent of WUS-CLV signaling altogether. The initial widening of SAMs observed in this study (fig. 2D) is similar to SEM observations of other model organisms and types of fasciation in that the SAM increases in size
14 while maintaining the dome morphology. This initial small size increase at the beginning of band fasciation in Glycine max was demonstrated by Tang and Skorpuska (1997). Comparable size differences between wild-type SAMs and those for multiple mutants of Arabidopsis thaliana less than two weeks after germination have also been shown by Clark et al. (1993) and Schoof et al. (2000). In the C. tembloriensis plants used for this study, this initial uniform size increase was much less drastic than those of later stages and only lasted for a short time (5-10 days) before beginning to show morphological characteristics indicative of ring fasciation. Although this initial widening stage of the SAM was subtle, the observed change in phyllotaxy to shorter internodes corresponded with a change in meristem regulation since phyllotaxy is a direct product of cell division patterns in the SAM (Traas 2013; Bykova et al. 2016). As initial widening continued in the C. tembloriensis plants of this study, SAMs often contributed very little to dome height, causing the apical dome to appear flatter (fig. 2F). This part of initial widening was indicative of the impending ring morphology for the apical dome, and it is possible that the central zone of the tunica began to slow division in preparation for differentiation at this time. The data in this study showed that normal phyllotaxy development continued to be affected by the increasing diameter of the SAM, generating more organs clustered around the apex. Due to the normal organ morphology of primordia at this stage, it is likely the change in phyllotaxy was an effect of phytohormone distribution within the enlarged peripheral zone. The spatial distribution and transport of auxin and cytokinin within the SAM is a well-known signal for primordia initiation in the peripheral zone in addition to meristem maintenance in the central zone (Stuurman et al. 2002; Fleming 2005; Traas 2013; Mandel et al. 2014; Schaller et al. 2015; Shi et al. 2018). This overlapping signaling suggests a particularly important role for auxin compared to other phytohormones involved with SAM fasciation in C. tembloriensis. Trichome production on the SAM central zone (fig. 3B) is a clear indication of progressive disruption in phytohormone signaling. Trichome production on the central zone of mutant Arabidopsis SAMs expressing fasciation has been shown by Schoof et al. (2000). The WUS-CLV feedback loop is the primary focus of their study, and its interactions with auxin signaling suggest once again that auxin may be a critical factor in development of SAM fasciation in C. tembloriensis. Mutant Petunia SAMs with trichome production following shoot termination have been shown by Stuurman et al. (2002). This supports the idea that trichome production and flattened dome morphology are associated with cell differentiation and reduced division in the upper tunica’s central zone. While SAM expansion along a single axis perpendicular to primary stem growth is indicative of band fasciation (White 1948; Tang and Skorupska 1997; Iliev and Kitin 2011; Singh et al. 2011), ring fasciation involves the meristematic proliferation of the entire SAM peripheral zone while the central zone drastically reduces division and differentiates into stem tissue (White 1948; Mertens and Burdick 1954; Majumdar 1955; Kundu and Rao 1960; Iliev and Kitin 2011; Choob and Sinyshin 2012). In the C. tembloriensis plants of this study, the meristematic peripheral zone appeared to proliferate past the SAM center to form a distinct ring morphology quite rapidly after the apparent differentiation of the central zone (fig. 3D). Phyllotaxy appeared to be altered yet again as the initiation of new primordia was extremely limited during ring formation. Cells within the corpus clearly remained meristematic during differentiation of the central tunica, allowing the apex to continually increase in diameter. The increase in apex diameter during ring formation was considerably greater than that of initial widening, indicating a breakdown in regulatory feedback signaling that normally restricts the size and shape of the pluripotent stem cell niche. Ring fasciation may be induced in SAMs by application of 2,3,5-triiodobenzoic acid (TIBA), a polar
15 auxin transport inhibitor, as demonstrated by previous investigations (Majumdar 1955; Iliev and Kitin 2011). Arabidopsis mutants for the PIN-FORMED1 (PIN1) gene are deficient in polar auxin transport but initiate a continuous peripheral ring organ following application of IAA to the SAM center (Reinhardt et al. 2003). The morphological and developmental similarities of those findings with the observed ring formation in the SAMs of C. tembloriensis expressing fasciation in this study further supports a central role for auxin signaling in ring fasciation. Following ring formation in C. tembloriensis SAMs expressing fasciation, longitudinal growth and lateral expansion of the meristematic ring began to produce a hollow tube or funnel morphology for the top of the primary axis (fig. 3D, 4B). Fasciated stems with a hollow morphology have been associated with ring fasciation for over a century (Compton 1911; White 1948; Mertens and Burdick 1954; Majumdar 1955; Sinjushin and Gostimsky 2006; Choob and Sinyushin 2012). In the C. tembloriensis plants in this study, the slow rate of longitudinal growth following ring formation coupled with the late onset of expression relative to that reported in other studies on SAM fasciation leads to a very small portion of hollow axis which is typically not apparent without a microscope. However, the classic hollow stem morphology associated with ring fasciation did appear to be present in C. tembloriensis for a brief period during development (fig. 3D, 4B). Initiation of leaf and floral primordia from the outer peripheral zone also appeared to resume following ring formation and produced a cluster of crowded primordia and young organs on the expanding apex (fig. 4B). The cluster of primordia on the ring’s periphery developed leaves and floral organs with abnormal curvature, partial fusion, or exposed ovules and appeared similar to SEM observations of abnormal leaves and floral organs on other plants expressing other forms of fasciation (Clark et al. 1993; Clark et al. 1995; Williams et al. 2005). Plants expressing fasciation have been reported in other studies to produce organs with abnormal curvature, fusion, or floral characteristics (Bowman et al. 1989; Putnam and Miller 2007; Iliev and Kitin 2011; Mandel et al. 2014), so it is not surprising that C. tembloriensis initiated leaves with abnormal curvature in the final enlargement stage. The single unfused carpels (fig. 4D, 4F) were unique due to their abnormal partially fused ovary wall that never fully enclosed the ovules, which is a significant departure from normal floral morphology in Clarkia. This morphological feature is further discussed below. As development of the C. tembloriensis apices in this study proceeded, friable callus tissue formed on the ring-like SAM surface. This is another indication of abnormal phytohormone signaling. Its effects on the central cavity and the adjacent organs suggest that the phenomenon was widespread throughout the enlarged ring-like SAM. The large, loosely-packed apical callus cells appeared nearly identical to previously reported SEM observations of callus tissue (Chaudhury and Qu 2000; Popielarska et al. 2006; Popielarska-Konieczna et al. 2008; Ng et al. 2016) and embryogenic tissue derived from callus (Sondahl et al. 1979). The development of callus or morphologically similar tissue on the surface of the ring-like SAM and on surrounding organ margins is not unique to fasciation in C. tembloriensis (Leyser and Furner 1992; Clark et al. 1995; Mandel et al. 2014). The marginal callus on surrounding leaf and floral primordia or young organs in the apical cluster of C. tembloriensis bears striking similarities in cell shape to stigmatic papillae in abnormal floral organs (Bowman et al. 1989; Clark et al. 1995; Eshed et al. 1999; Favaro et al. 2003; Mandel et al. 2014). The frequent appearance of abnormal flowers in C. tembloriensis expressing fasciation in the final enlargement stages also suggests that the tissue may be ectopic stigma papillae like that demonstrated in several studies that focused on abnormal floral development (Bowman et al. 1989; Clark et al. 1995; Eshed et al. 1999; Favaro et al. 2003), but the proliferation of this tissue appears to result in the formation of 16 large white friable callus growths on the base of abnormal organ margins such as the curved leaf described above (fig. 1D). These large friable callus growths found on abnormal organ margins are morphologically identical to calli demonstrated in other studies (Chaudhury and Qu 2000; Cheng et al. 2013; Ikeuchi et al. 2013; Ng et al. 2016), so it may be that abnormal cell identity signaling causes stigmatic papillae to develop on organ margins first, and then this stigmatic tissue is either covered by undifferentiated callus during proliferation at the apex or grows intermingled with callus as shown by Clark et al. (1995). Callus development typically results from excess availability of certain nutrients (Reed et al. 2013) or phytohormone signaling (Chaudhury and Qu 2000; Popielarska et al. 2006; Mitras et al. 2009; Cheng et al. 2013). Exogenous application of auxin and cytokinin to various plant species induces callus formation and the ratio of auxin to cytokinin applied determines the type of callus generated (Ikeuchi et al. 2013). The development and proliferation of callus tissue replacing the surface of the ring- shaped SAM and filling the central cavity of C. tembloriensis plants expressing fasciation is therefore indicative of abnormal phytohormone signaling during the final stages of apex enlargement, and once again suggests that auxin and cytokinin are critical factors in these morphological changes. After the apical periphery of the C. tembloriensis plants in this study was crowded with organ primordia and as callus tissue began proliferation, phyllotaxy appeared to undergo another change to dramatically slow or stop initiation of new leaf and floral primordia (fig. 4D, 4F). Organs at the apex developing with abnormally warped, fused, stunted, or pin-like morphology were likely this way due primarily to abnormal phytohormone signaling throughout the enlarged apex as observed in Arabidopsis thaliana pin1 mutants by Vernoux et al. (2000). The crowded spatial distribution may have an effect on phyllotaxy and individual organ shape as well, demonstrated in Helianthus by Hernandez and Green (1993) and supported by many additional observations (Green 1999). Although there was little longitudinal growth, the CWT apices increased their diameter significantly during the final enlargement and callus proliferation stages to become many times the size of a normal CC1 apex by the end of a typical growing season (fig. 4E-F, 5A). This facilitated the development of an apical leaf and flower cluster since more surface area was available at the site of initiation in a short length of stem. It is also indicative of an abnormally regulated network for maintenance of the stem cell niche and subsequent identity signaling towards the periphery, both of which have been shown to be affected by auxin and cytokinin concentration in Arabidopsis (Zhao et al. 2010). The final stages of SAM enlargement in the C. tembloriensis plants expressing ring fasciation in this study included callus proliferation toward the central cavity, which changed the apparent morphology by filling part of the cavity. In most cases this meant a slight change in morphology from a longer tube or funnel cavity to a shallower concave bowl shape filled with friable callus (fig. 5C) which tended to be obstructed by growth of surrounding organs (fig. 5A). In some cases, this resulted in the development of a flat or convex mass of callus in the central portion of the apex (fig. 4F, 5B). The variation in callus proliferation is not unusual considering the state of phytohormone regulation, or lack thereof. It is well-known that phytohormones are responsible for callus formation (Chaudhury and Qu 2000, Popielarska et al. 2006, Cheng et al. 2013). For decades, callus-inducing media in controlled studies like that of Sondahl et al. (1979) has included synthetic and isolated phytohormones like NAA and kinetin. Accumulation of auxin promotes initiation of leaf and flower primordia by signaling cells in the peripheral zone to differentiate, demonstrated by Reinhardt et al. (2003) and supported by other research (Heisler et al. 2005; Zhao et al. 2010). Cytokinin has even been shown to be under direct negative control 17 by WUS in Arabidopsis (Leibfried et al. 2005). The activity of both phytohormones in SAMs has also been shown to converge via genetic regulatory framework by Zhao et al. (2010) and other researchers (Schaller et al. 2015; Shi et al. 2018). This interaction is crucial to several aspects of meristem maintenance and Besnard et al. (2014) demonstrated that it directly regulates the timing of organ initiation. Variations in distribution or concentration of phytohormones can have a variety of effects on the size, morphology, and generative capability of calli (Mitras et al. 2009, Ikeuchi et al. 2013), making it incredibly difficult to associate morphological characteristics of callus tissue with specific molecular pathways without some method of visualizing or quantifying phytohormone concentrations during formation. LM Observations of SAM Development Normal SAM anatomy for nearly all angiosperms is characterized by cytohistological zones where the more general corpus and tunica superimpose the organizing center, rib meristem, peripheral zone, and tunica layers L1, L2, and L3 (Beck 2010; Schaller et al. 2015). In vegetative SAMs of most herbaceous dicotyledonous angiosperms with spiral phyllotaxy such as Arabidopsis thaliana, the organizing center refers to the pool of pluripotent stem cells whose size and shape is regulated by expression of WUS and CLV3 or respective orthologs (among other partially redundant genes) and normally maintains a round to elliptical shape in longitudinal section ranging from one to several cells wide as demonstrated in Arabidopsis by Shi et al. (2018) and many other past publications (Clark et al. 1995; Carles et al. 2005; Beck 2010; Mandel et al. 2014; Schaller et al. 2015; Xu et al. 2015). The C. tembloriensis plants observed in this study appeared to adhere to this anatomical pattern throughout development in normal CC1 apices and until the end of initial widening in CWT plants expressing fasciation (fig. 6A-F). Initial widening appeared to affect all central layers of cytohistological zonation uniformly, increasing the number of cells by a small but noticeable margin laterally while maintaining a consistent height (fig. 6B). This is typical for the beginning stages of SAM fasciation in Arabidopsis and Zea mays for multiple types of fasciation (Schoof et al. 2000; Williams et al. 2005; Mandel et al. 2014; Pautler et al. 2015; Xu et al. 2015; Je et al. 2018). At the end of initial widening in C. tembloriensis, the central cytohistological zones appeared to continue the pattern of uniformly increasing diameter in addition to a small height increase for the L3 tunica layer and organizing center (fig. 6F). This is also not out of the ordinary for plants expressing fasciation in initial stages of diameter increase, though band fasciation entails expansion along a single linear, lateral axis rather than expanding radially as shown in Populus hybrids, Arabidopsis thaliana, and Zea mays (Nilsson et al. 1996; Schoof et al. 2000; Fletcher 2001; Williams et al. 2005; Mandel et al. 2014; Pautler et al. 2015; Yang et al. 2015; Je et al. 2016). The observed difference in cytoplasmic activity between the center of the organizing center and more peripheral corpus tissue is also normal for SAMs due to the slower rate of division in these cells required to maintain a smaller population as described for Arabidopsis mutants by Clark et al. (1995) and other researchers (Iliev and Kitin 2011; Pautler et al. 2015). One outstanding anatomical change occured in the center of the L1 tunica layer such that cytoplasmic activity was decreased, and vacuolar space took up a noticeable portion of the cell volume. The anatomical similarity of the central L1 layer to differentiating epidermal cells is very likely an indication that the central SAM surface has begun differentiation and is nearing the point of trichome development and peripheral outgrowth. This is further supported by the developmental context of an abnormally regulated SAM.
18
Soon after the initial widening stage in the experimental C. tembloriensis plants of this study, the SAM corpus appeared to rapidly increase in both diameter and height (fig. 8B). While an enlarged corpus region is a common observation in SAMs expressing fasciation for model plants such as Arabidopsis thaliana (Schoof et al. 2000; Kaya et al. 2001; Zhao et al. 2010) and Echinocereus reichenbachii (Boke and Ross 1978), the cytohistological zones observed in this experiment demonstrated several anatomical abnormalities. Tunica organization did appear to retain an L1 layer, though a flat or undulating surface producing trichomes changed the layer’s pattern, and callus production appeared to be initiated around this time, distorting the zone’s appearance even further (fig. 7B). The boundary between the L2 and L3 layers was not easily distinguishable in apices in early ring formation, but the lower portion of the L3 layer appeared to comprise the majority of the enlarged corpus. The center of the enlarged corpus had reduced staining compared to the apparent periphery, which likely indicated this was the boundary of the lower L3 layer with the peripheral zone. The organizing center was much larger than normal and could be identified by small cell sizes relative to surrounding corpus and rib meristem tissue and a central region with heavy cytoplasmic activity. This indicates a breakdown in regulation of cell division rates within the organizing center because the organizing center should normally maintain a slower rate of division than surrounding corpus tissue as shown previously in Arabidopsis (Clark et al. 1995; Iliev and Kitin 2011; Pautler et al. 2015). The rib meristem also had an increased diameter and produced parenchyma cells that were approximately twice as wide as those produced by normal SAMs of a similar age that did not appear to elongate to the same degree, and that had more intracellular space occupied by vacuoles (fig. 8B). This rib meristem morphology indicated that abnormal phytohormone concentrations may be present in this tissue due to the potential effects of auxin and cytokinin on cell division patterns, similar to observations of Arabidopsis mutants for multiple genes involved in auxin transport and cytokinin signaling by Zhao et al. (2010), among other findings (Flemming 2005; Heisler et al. 2005; Barbier de Reuille et al. 2006; Cheng et al. 2013; Besnard et al. 2014). Lastly, C. tembloriensis SAMs in the early ring formation stage of expressing fasciation had a much thicker vascular strand than normal SAMs, which was typically not visible so close to the apex (fig. 8A-D). Increased production and delayed development of vascular tissue is a characteristic often associated with fasciation in other plants such as Pisum sativum, Fraxinus excelsior, Populus hybrids, Betula pendula, and more (Nilsson et al. 1996; Sinjushin and Gostimsky 2006; Mitras et al. 2009; Iliev and Kitin 2011), and so it is not surprising to see abnormal vascular development occurring in C. tembloriensis plants expressing ring fasciation. During late ring formation in the C. tembloriensis plants of this experiment, SAMs expressing fasciation continued to increase the size of cytohistological zones and many organs may be seen clustered at the periphery (fig. 8D). The L1 tunica layer appeared to continue developing into callus tissue on the surface of the apical ring where a thick integument, small cell size, heavy staining of cytoplasm, and inconsistent cell shape indicated unregulated cell proliferation in all but the centermost L1 cells (fig. 7D). A thick integument on the central surface of the ring-shaped SAM further supports the idea that abnormal cell identity signaling was associated with the L1 tunica layer differentiating into epidermal tissue prior to callus development. Abnormal cell identity signaling may be set in motion by changes in phytohormone concentration, which is the basis of callus formation in studies like that of Ikeuchi et al. (2013) while several other researchers offer experimental support for involvement of phytohormones in abnormal cell identity signaling (Stuurman et al. 2002; Mandel et al. 2014; Schaller et al. 2015). Underlying this layer is an L2 layer with little cytoplasmic activity
19 compared to surrounding cells. This is uncommon for cells of the L2 layer which share a similar anatomy with the underlying L3 layer in normal SAMs (Fleming 2005; Choob and Sinyushin 2012) and may be indicative of changes to genetic expression levels in the center of the L2 and L3 layers as demonstrated in Arabidopsis by Vernoux et al. (2011). This suggests that the central tunica layer may either regain a pluripotent state during ring formation or that the developing callus tissue comprises a new cytohistological zone atop the L1 layer. In the C. tembloriensis plants of this study, the L3 tunica layer’s darkly staining cytoplasm throughout the cells in the upper portion of the SAM indicates continued cell division in the outer corpus despite signs of differentiation in the center of the upper layers (fig. 7D, 8D). This was a likely contributing factor to both peripheral ring formation and development of the cluster of young organs found around the periphery during late ring formation and the beginning of final enlargement. Underlying this zone of rapid division was an area with lighter staining, indicating a much slower rate of division for the innermost portion of the L3 layer. This is normal for the center of the corpus in SAMs due to the accelerated growth rate required to occupy the larger space as described previously in Arabidopsis (Clark et al. 1995) and other model plants (Beck 2010; Iliev and Kitin 2011; Choob and Sinyushin 2012; Pautler et al. 2015), but the cell size and cell file orientation closely resembled that of rib meristem tissue. The organizing center of the C. tembloriensis plants in this study remained far beneath the ring-like surface of the SAM during late ring formation and appeared to be more easily distinguishable due to the difference in cell size and staining compared to the surrounding rib meristem and lower L3 layer (fig. 8D). The organizing center’s proximity to a more differentiated rib zone supports the idea that longitudinal stem growth is minimal by this point in development and the presence of raphides so close to the organizing center reinforces this further. Stem tissue interior to the young vascular strands shows signs of breaking down from insufficient resin infiltration (fig. 8D), but the adequate preservation of the rib meristem and corpus tissue make the anatomical origin of this insufficient infiltration unclear. Cell size in plant tissues may be influenced by a variety of environmental, phytohormonal, and genetic factors (Clouse et al. 1996; Kauschmann et al. 1996; Werner et al. 2003; Flemming 2005; Fukuda et al. 2008; Shi et al. 2018), and it is common for friable callus tissue to reach larger than normal cell sizes in plants such as Actinidia deliciosa as demonstrated by Popielarska et al. (2006) and others (Ng et al. 2016). The high proportion of vacuolar space found in C. tembloriensis callus tissue is also not unusual for cells of abnormally large size as shown for Boesenbergia rotunda callus by Ng et al. (2016). The complex cell wall shape displayed by some callus cells is likely due to a combination of the effects of osmotic forces during preservation, spatial limitations during proliferation, abnormal phytohormone signaling, and altered gene expression. However, determining the extent to which each factor affects the shape of callus cells is beyond the scope of this study. The interior transition of callus tissue to parenchyma reinforces support for callus development directly over the top of an existing SAM. The younger callus tissue described above as small, loosely packed cells with dense cytoplasm generally found on abnormal apical organs clearly showed signs of active division. This suggests that rapid proliferation of the callus tissue occurred while in an undifferentiated state, whereas the larger cells found in this younger callus tissue showed an intermediate state of differentiation. Their round shape and expanding vacuoles indicate that the size increase of callus cells seen in SEM observations was associated with development of mature callus tissue that proliferated at a slower rate, if at all. Abnormal Floral Features 20
On C. tembloriensis plants expressing fasciation in this study, single unfused carpels that surrounded an enlarged ring-like SAM or apical callus tissue and abnormal flowers initiated from the apical callus periphery appeared to be the result of abnormal phytohormone signaling. This speculation is reasonable due to the known effect of environmental conditions on phytohormone signaling (Man et al. 2011) and subsequent effect of phytohormone signaling on genetic expression that regulates organ development (Heisler et al. 2005; Prigge et al. 2005; Beck 2010; Vernoux et al. 2011). Favaro et al. (2003) provide strikingly similar observations to the unfused carpels with sepals of transgenic mutant Arabidopsis plants identified as curled carpeloid sepals with ectopic ovules. The relatively consistent morphological features of the single unfused carpels observed in this study on C. tembloriensis further suggests that cell identity was affected by abnormal molecular signaling associated with fasciation. Flower development involves a complex array of phytohormonal and genetic framework overlapping with the regulatory networks in meristem maintenance (Bowman et al. 1989; Clark et al. 1995; Laux et al. 1996; Favaro et al. 2003; Heisler et al. 2005; Williams et al. 2005; Cucinotta et al. 2014; Mandel et al. 2014; Pautler et al. 2015). This molecular signaling overlap would explain the early initiation of normal flowers in C. tembloriensis SAMs expressing fasciation up to ring formation, when it is likely that abnormal phytohormone concentrations at the apex would become more widespread. The initiation of ovules on the side of unfused carpels closest to the expanding apex also supports this idea because ovule initiation is greatly influenced by auxin concentration (Cucinotta et al. 2014). In C. tembloriensis plants observed in this study, abnormal flowers initiated from the apical callus periphery had some distinctive differences from the single, unfused carpels surrounding apices following the appearance of an enlarged ring-like SAM or callus tissue. Among these differences, perhaps the most notable was the abnormal style and stigma appearing in the approximate center of these organs adjacent to an existing unfused carpel ovary, clearly indicating a floral organ identity. The callus tissue appeared to facilitate fusion to surrounding carpels in addition to increasing the overall size of the apex. Although the stigma’s surface was initially covered in younger callus tissue like that found on surrounding organ margins, later development showed more mature stigma tissue similar to observations in other plants (McConnell and Barton 1998; Eshed et al. 1999; Favaro et al. 2003; Mandel et al. 2014). After the abnormal style underwent elongation, surrounding unfused carpels with unfused outer wall morphology collectively appeared similar to an abnormal flower at the apex, further suggesting a phytohormonal link between disruption of SAM maintenance and floral organ development. The underlying SAM showing very few signs of ongoing division indicated that the callus tissue became largely responsible for final enlargement of the apex beyond this point and that more growth may be devoted to development of the abnormal floral organs rather than increasing the stem diameter from the organizing center. The development of ovules in such a linear pattern on abnormal organs not only reinforces a carpel identity and abnormal phytohormonal signaling, it could also provide insight into the abnormal transport patterns of phytohormones known to regulate ovule formation, such as auxin and cytokinin (Heisler et al. 2005; Cucinotta et al. 2014; Mandel et al. 2014). The formation of normal ovules near organ boundaries also implicates the involvement of brassinosteroids, which play a key role in organ boundary formation and interact with auxin signaling (Kauschmann et al. 1996; Vert et al. 2008; Gendron et al. 2012). Observations of Growth in the Field
21
While natural occurrence of fasciation in the wild was not observed during field collections of July 2016, the population thought to have the highest likelihood of expressing fasciation (Crocker Canyon) was found to be heavily impacted by herbivore activity and the timing of the visit was one to two months following the end of flowering time and El Nino rainfall when fasciation would be least challenging to identify. In addition, some populations were observed to have dried stalks reaching 1-1.5 m in height with relatively thick (up to 1 cm) stems. Although the apex of the primary axes of these dried field plants was not indicative of fasciation, such vigorous growth was uncommon in chamber-grown plants and serves as an excellent indication that the environmental conditions were conducive for fasciation in natural populations of C. tembloriensis. However, it may be the case that only certain populations (i.e. Crocker Canyon) carry the genetic predisposition for its expression in their gene pool. That would make C. tembloriensis a model organism of special interest to researchers investigating genes involved in SAM maintenance.
Conclusion
The expression of SAM fasciation in Clarkia tembloriensis entails 3 morphological stages of development including initial widening, ring formation, and final enlargement. Although surface morphology and the underlying anatomy of the SAM during initial widening adheres to a classic model of fasciation with easily defined cytohistological zonation, ring formation brings about changes that diverge from normal development in a progressive manner. The environmental stimulus for fasciation used in this study in combination with an apparently cascading disruption in meristem size maintenance suggests that phytohormones such as auxin, cytokinin, and brassinosteroids are essential for expression of ring fasciation in C. tembloriensis. The specific molecular pathways to achieve ring morphology and abnormal organ development in C. tembloriensis are beyond the scope of this study, but comparisons between these results and the literature suggest that expression domain of WUS is affected as a result of the experimental treatment. The appearance of callus tissue on apices expressing fasciation reinforces support for both abnormal phytohormone signaling and disrupted regulation of undifferentiated tissue proliferation. Overall, this study provides compelling evidence documenting the development of SAMs expressing ring fasciation in Clarkia tembloriensis and enhances the existing scientific groundwork for understanding the nature of fasciation in plants, a growth condition with an existing history and considerable potential for application in agriculture.
22
Figures
A B
C D
Figure 1. Digital photographs of selected morphological abnormalities associated with fasciation during initial observations. Symbol Key: = normal round shoot and flat shoot expressing fasciation, = primary stem, = new shoots emerging from apex, = stunted leaves, = leaves with abnormal curvature fused to young secondary stems, = new shoot growing in a loop, = friable tissue growth on interior edge of a curved leaf with no central vein.
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Figure 2. SEM images of early SAM development in normal CC1 plants (left) and CWT plants expressing fasciation (right). SAM central zone and surrounding organs are indicated by color. Bar = 100 µm. Apices A-F were fixed at 27, 28, 31, 32, 42, and 43 DAT, respectively. Color Key: gold = SAM surface, green = leaf primordium, red = floral meristem or young flower
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Figure 3. SEM images of mid-stage SAM development in normal CC1 plants (left) and CWT plants expressing fasciation (right). SAM surface and surrounding organs are indicated by color or symbol. Bar = 100 µm. Apices A-D were fixed at 49, 50, 49, and 56 DAT, respectively. Color key: gold = SAM surface, green = leaf primordia, red = floral meristems or young flowers. Symbol Key: = trichomes on the SAM surface.
25
Figure 4. SEM images of late SAM development in normal plants (left) and those expressing fasciation (right). The SAM surface, callus tissue, and surrounding organs left at least partially intact are indicated by color. Bar = 100 µm. Apices A-F were fixed at 52, 50, 55, 56, 58, and 59 DAT, respectively. Color Key: gold = SAM surface, cyan = callus tissue, green = organs developing with normal and abnormal leaf morphology, red = floral meristems, normal flowers, and abnormal flowers, orange = abnormal pin-like organs. Symbol Key: = trichomes on the SAM surface, = exposed ovules.
26
Figure 5. SEM images of apical callus development on extremely enlarged SAMs expressing fasciation in late development. Callus tissue and surrounding organs left at least partially intact are indicated by color. Bar = 100 µm. Apices A and B are from CWT plants fixed at 59 DAT and apex C is from an observational group of CCP plants grown under similar chamber conditions and fixed late in development. Color Key: cyan = callus tissue, green = young leaves, red = abnormal flowers. Symbol Key: = exposed ovules.
27
A B
C D
E F
Figure 6. LM images of early SAM development in normal CC1 plants (left) and CWT plants expressing fasciation (right). Bar = 100 µm. Apices A-F were fixed at 27, 32, 31, 32, 42, and 39 DAT, respectively. Color Key: red = L1 central zone, orange = L2 central zone, green = L3 central zone, gold = organizing center, violet = rib meristem. Symbol Key: = leaf primordia, = floral primordia.
28
A B
C D
Figure 7. LM images of mid-stage SAM development in normal CC1 plants (left) and CWT plants expressing fasciation (right). Bar = 100 µm. Apices A-D were fixed at 49, 47, 52, and 50 DAT, respectively. Symbol Key: = leaf primordia, = floral primordia, = young callus tissue, = trichomes on apical surface, = ring-shaped periphery, = lower tunica layers with dense cytoplasm.
29
A B
C D
R Figure 8. Low-magnification LM images of mid-stage SAM development in normal CC1 plants (left) and CWT plants expressing fasciation (right). Bar = 100 µm. Apices A-D were fixed at 49, 47, 52, and 50 DAT, respectively. Symbol Key: = leaf primordia, = floral primordia, = young callus tissue, = trichomes on apical surface, = ring-shaped periphery, = enlarged corpus, = provascular strands, = outer edges of rib meristem, = raphide.
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A
B
Figure 9. LM images of callus tissue development in older CCP lateral shoot (top) and CWT apex (bottom) expressing fasciation. Bar = 100 µm. Apices A-B were fixed at 73 DAT and 59 DAT, respectively. Symbol Key: = mature callus, = young callus tissue, = developing callus tissue, = cell anatomy gradient from enlarged and irregular callus to normal interior parenchyma.
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Figure 10. SEM images of abnormal floral features in CWT apices expressing fasciation late in development. Bar = 100 µm. Apices A-B were fixed at 59 and 56 DAT, respectively. Color Key: red = abnormal floral organs including carpel ovaries, style, and stigma, cyan = callus tissue on the apex. Symbol Key: = exposed ovules. 32
Figure 11. LM image of abnormal floral features in CWT apices expressing fasciation late in development. Bar = 100 µm. Apex was fixed at 59 DAT. Symbol key: = exposed ovules, = abnormal stigma.
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A
B
Figure 12. LM images of ovule (top) and abnormal stigma (bottom) in CWT apices expressing fasciation late in development. Bar = 100 µm. Symbol Key: = layer of developing callus tissue on the stigma.
34
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Appendices
Appendix 1 – Conceptual Diagram of SAM development in Clarkia tembloriensis
Appendix 1. Conceptual diagram of SAM development in CWT apices expressing fasciation showed without development of a centralized abnormal flower on the apex (top) and with development of a centralized abnormal flower on the apex (bottom). Red shading indicates areas where callus may develop on the surface.
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Appendix 2 – Shoot Apex Removal Experiment The influence of apical meristem removal, the cp gene, and abnormal environmental conditions (i.e. available water and nutrients) on plant growth was investigated in chamber- grown CCP and CWT plants with respect to height, lateral branch development, and recovery of apical dominance. Plants were spatially randomized within each chamber using a random number generator, then grown for approximately 3 months. Apices designated for such treatment were removed once during a 3-month growing period (details by replicate below) by cutting through the stem with a scalpel down to the first or second distinguishable node below the apex. Remaining shoot apices were then fixed in FAA and used for light microscopy. At the end of the 3-month growth period, the final height was measured and overall plant architecture was scored. The effect of apex removal was assessed by comparing measurements between removal treatment groups for plant height and response, where response was recorded as either SB for enhanced lateral branch development, WT for wild-type primary shoot growth (from highest node below cut, if removed), or F for fasciated primary shoot growth (from highest node below cut, if removed). When the highest node on the primary shoot did not accelerate its growth and recover apical dominance, instead promoting lower lateral branches to grow in excess of 10 cm, the response was recorded as enhanced lateral branch development. The effect of limiting water and nutrients was also assessed by varying the number of plants per pot (2 per pot vs. 10 per pot) and the amount of available water (non-limiting vs. 1 cup per pot per day). These environmental resources were combined into a single treatment category designated as non-limited resources (2 plants per pot, watering when topsoil dries) or limited resources (10 plants per pot, 1 cup of water per pot per day). Therefore, the 2 experimental groups (CWT and CCP) received 2 treatments (non-limited/limited resources and intact/removed shoot apex), giving 1 of 3 possible plant architecture responses (SB, WT, F). There were 3 replicates of the experiment due to available chamber space. The first replicate included 40 CWT and 40 CCP plants, where half of each group was given the non- limiting resource treatment. When all plants reached at least 5 inches (12.7 cm) in height (7 weeks after transplanting), apices were removed as described above from 20 CWT and 20 CCP plants where half of each experimental group was part of the limited resource treatment. The second replicate included 40 CWT and 18 CCP plants, where half of CWT plants and 8 CCP plants were given the non-limiting resource treatment. At 5 weeks after transplanting, shoot apices were removed as described above from 20 CWT and 9 CCP plants where 10 CWT and 5 CCP plants were part of the limited resource treatment. The third replicate included 40 CWT and 40 CCP plants, with half of each group given the non-limiting resource treatment. At 5 weeks after transplanting, apices were removed as described above from 20 CWT and 20 CCP plants where half of each experimental group was given the limited resource treatment. After plants in each replicate were grown for approximately 3 months, response and height data was collected and differences between experimental groups were analyzed in R Studio. Multiple logistic regression analyses were used to assess whether presence of the cp gene, non-limiting environmental resources, or apex removal were significant predictors of fasciation or enhanced lateral shoot growth. Multiple linear regression analysis was used to assess whether presence of the CP gene, water and nutrient availability, or expression of fasciation were significant predictors of primary shoot height in plants with apices left intact. Investigation of plant growth responses to presence of the cp gene, water and nutrient availability, and shoot apex removal resulted in several statistically significant relationships
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(Supplemental Table 1). Multiple logistic regression analysis indicates that the cp gene, resource availability, and apex removal are all statistically significant predictors of fasciation (p < 0.001). Non-limiting resources and the cp gene both appear to increase the chance of fasciation. Removal of the entire SAM did not frequently result in the lateral shoot closest to the cut growing rapidly in its place and becoming fasciated (Supplemental Table 2), and multiple logistic regression analysis supports that it decreases the chance of expressing fasciation in the new pseudo-SAM. Analysis of the enhanced lateral growth response variable indicates that resource availability is the only statistically significant predictor variable (p < 0.001), and that it has a positive influence on lateral shoot growth. Surprisingly, apex removal was not found to be a significant predictor (p = 0.988) even though all plants with enhanced lateral growth were part of the apex removal treatment (Supplementary Table 2). Investigation of plant height in response to presence of the CP gene, water and nutrient availability, and fasciation yielded unsurprising results (Supplementary Table 1). Multiple linear regression analysis indicates that the CP gene is a statistically significant predictor variable (p < 0.001) with a negative effect on height and that resource availability is a statistically significant predictor variable (p < 0.001) with a positive effect on height. Somewhat surprisingly, fasciation was not found to be a statistically significant predictor variable for height (p = 0.053). Overall model fit was good (p < 0.001) in all but the multiple logistic regression for branching response, which is not statistically significant (p = 0.989). Investigation of plant growth responses to presence of the CP gene, water and nutrient availability, and shoot apex removal yielded largely expected results (Supplementary Table 1). The likelihood of expressing fasciation was increased by the CP gene and resource availability, an expected finding that supports the observations of Smith-Huerta and Huerta (2015 poster). The effect of resource availability is of particular interest because it provides additional support for methods used to stimulate fasciation in plants for microscopic SAM observation. Similar effects of nutrient availability on plant growth have been noted previously (White 1948, Iliev and Kitin 2011, Reed et al. 2013). Since it has been suggested that physical damage (i.e. herbivory) to the SAM can stimulate fasciation (White 1948, Alka and Ansari 2013, Geneve 1990), the effect of apex removal was also included in this analysis. In agreement with Ansari and Daehler (2011), results suggest that apex removal effectively prevents expression of fasciation at the main stem and therefore that environmental forces damaging the apex like herbivory are not likely to play a role in fasciation in C. tembloriensis. The positive effect of resource availability on lateral branching is not surprising, as more resources would be allocated to vegetative growth. However, the apparent lack of effect on lateral branching from apex removal was very surprising. While not all plants with apices removed showed enhanced lateral branching, all those that did show enhanced lateral branching were part of the damaged group. This model was also associated with high standard error and poor overall fit (table 1), suggesting something in the data may be confounding these results. It is known that removing the SAM of a plant reduces inhibition of lateral bud growth and allocates more resources to lateral shoot growth (Leyser 2005, Irwin and Aarssen 1996). Although multiple logistic regression analysis did not show this effect to be significant, the raw data appear to be consistent with the literature. Investigation of plant height in response to the CP gene, resource availability, and fasciation yielded interesting results. Smith-Huerta and Huerta (2015 poster) demonstrated that the CP gene negatively affects height in C. tembloriensis under well-watered conditions. Because
46 the CP gene has also been shown to increase the frequency of expressing fasciation in C. tembloriensis (Smith-Huerta and Huerta 2015 poster), and fasciation is associated primarily with an increase in SAM diameter (White 1948, Iliev and Kitin 2011, Leyser and Furner 1992, Tang and Skorupska 1997, Singh et al. 2011, Bykova et al. 2016, Mandel et al. 2014), it was hypothesized that the CP gene would have a negative effect on plant height. In contrast, table 1 shows that the CP gene is a positive predictor for plant height. It is possible that including the resource-limited group in the analysis accounts for differences between these results and those of Smith-Huerta and Huerta (2015 poster), as only their well-watered group provided statistically significant differences. This would suggest that the effect of the CP gene on plant height is dependent on resource availability. Resource availability was expected to have a positive influence on height, and results were consistent with this. Fasciation was expected to have a negative influence on plant height because proportionally more resources could be allocated to stem thickening and initiation of new organs. Unexpectedly, fasciation was not a statistically significant predictor for height at a 95% confidence interval (Supplementary Table 1). While this finding contrasted with the prediction, it is not incredibly surprising that cell proliferation within the SAM occurs at a high enough rate to continually add to both the height and thickness of the stem. Many researchers have reported that disruptions in the WUS-CLV regulatory signaling pathway leads to increased cell division and SAM size in Arabidopsis (Clark et al. 1993, Williams and Fletcher 2005, Mandel et al. 2014, Shi et al. 2018). Porbeni and Fawole (2013) found increased height in Cowpea expressing stem fasciation compared with wild-type growth, additionally noting delayed maturity and decreased seed yields in fasciated plants. This is much different from what has been observed in C. tembloriensis and demonstrates that the effect of fasciation on height (and reproduction) is likely a complex, multifactorial issue partially dependent on plant architecture.
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Supplemental Table 1. Results of Statistical Analyses for Apex Removal Predictor Effect Response Test Significance cp gene + Fasciation log *** Resource Availability + Fasciation log *** Removal - Fasciation log *** Model (F) - Fasciation log *** cp gene - Enhanced Lateral Growth log NS Resource Availability + Enhanced Lateral Growth log *** Removal + Enhanced Lateral Growth log NS Model (SB) - Enhanced Lateral Growth log NS cp gene - Height lin *** Resource Availability + Height lin *** Fasciation + Height lin NS Model (Height) + Height lin *** *Abbreviations: Fasciation (F), enhanced lateral growth (SB), negative predictor effect (-), positive predictor effect (+), multiple logistic regression (log), multiple linear regression (lin). Statistical significance: 99.9% confidence (***), 99% confidence (**), 95% confidence (*), not significant (NS). Supplemental Table 2. Summary of Apex Removal Experiment Data CWT CCP Plant Growth Responses LR NLR LR NLR ND AR ND AR ND AR ND AR Count 0 0 18 2 8 1 23 0 F Frequency (%) - - 60.0 6.0 32.0 4.0 100.0 - Height (cm) - - 107.3 69.8 65.4 61.3 76.4 - Count 0 21 0 28 0 13 0 25 SB Frequency (%) - 70.0 - 93.3 - 52.0 - 100.0 Height (cm) - 26.4 - 41.6 - 23.5 - 35.2 Count 30 9 12 0 17 11 0 0 WT Frequency (%) 100.0 30.0 40.0 - 68.0 44.0 - - Height (cm) 63.4 31.6 104.1 - 42.5 44.6 - - Count 30 30 30 30 25 25 23 25 All Height (cm) 63.4 28.0 106.0 43.5 49.8 34.3 76.4 35.2 *Abbreviations: Fasciation (F), enhanced lateral branching (SB), wild-type growth (WT), Crocker Canyon plants not expressing the cp trait (CWT), Crocker Canyon plants expressing the cp trait (CCP), limited resource treatment (LR), non-limiting resource treatment (NLR), no damage (ND), apex removal (AR).
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