Evaluating Ethylene Sensitivity Using Mature Plant Screens and the Seedling Hypocotyl Response

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Nichole Frances Edelman, B. A.

Graduate Program in Horticulture and Crop Science

The Ohio State University

2013

Master's Examination Committee:

Dr. Michelle L. Jones, Advisor

Dr. Pablo Jourdan

Dr. Claudio Pasian

Copyrighted by

Nichole Frances Edelman

2013

Abstract

Ethylene (C2H4) is a gaseous hormone produced by plants in response to environmental stress and during growth and development. Sensitive seedlings exposed to ethylene gas will exhibit an exaggerated apical hook, thickened hypocotyl, and reduced hypocotyl elongation. Collectively these symptoms are known as the triple response, and have been used as a screen to identify ethylene mutants. Exposure to ethylene gas at the mature plant stage can induce flower, bud, or leaf abscission, flower senescence, leaf chlorosis

(yellowing), or leaf epinasty (downward curvature). Sensitivity can vary between species and by developmental stage or tissue (flower vs leaf). Ethylene can damage plants during production, shipping, and retailing. The objectives of this research were 1) to determine if the seedling hypocotyl elongation screen could be used to predict the sensitivity of mature plants at the marketable stage; 2) to identify ethylene sensitivity differences

(levels of sensitivity and symptoms) between accessions within Solanaceae; and 3) to identify ethylene sensitivity differences at different developmental stages (seedling, juvenile, and mature plants). Seedlings were germinated on filter paper saturated with 1- aminocyclopropane-1-carboxylic acid (ACC) and sensitivity was determined based on the hypocotyl lengths relative to the control (0 µM ACC). Mature plants were treated with 10 µL·L-1 ethylene gas for 24 hours, and the severity of the response was used to

ii classify ethylene sensitivity. The first experiment evaluated 18 bedding plant species while the second evaluated 40 Solanaceous accessions. A classification of low, medium or high sensitivity to ethylene was assigned to plants at both developmental stages. The results showed that the seedling hypocotyl elongation screen did not consistently predict mature plant sensitivity. Ethylene sensitivity varied between species and cultivars within the Solanaceae family, and sensitivity also varied by developmental stage. Information from this research can be used by growers to identify nonresponsive or low ethylene sensitive varieties for long-distance shipping and to identify medium to highly sensitive varieties that would benefit from treatment with ethylene inhibitors (1-MCP). Plant breeders and scientists can also use this information to identify breeding lines with lower ethylene sensitivity.

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Acknowledgments

I would first like to thank my advisor, Dr. Michelle Jones without whose amazing guidance and support none of this would have been possible. I would also to thank the other members of my student advisory committee, Dr. Pablo Jourdan and Dr. Claudio

Pasian for their suggestions on research and critical review of the chapters. I would like to thank the members of my lab including Laura Chapin, Eileen Ramsay, Shaun

Broderick, and previous member Cassi Sewell for support, technical and statistical assistance, and review of my chapters. I would also like to thank Dr. David Francis for statistical advice.

I would like to acknowledge the Ohio Agricultural Research and Development

Center (OARDC), and The Ohio State University D. C. Kiplinger Floriculture

Endowment for funding and supporting this research. I would also like to acknowledge

Ball Horticulture Company and Green Circle Growers for providing seeds and cuttings used in the experiments.

Last, I would like to thank my parents and friends for their support and love, and to those who encouraged me in my career goals and furthering my education. With their help and support I had to courage to keep pursuing my goals.

Vita

1999-2004 ...... Euclid High School

2004-2005 ...... Notre Dame College

2005-2008 ...... B. A. Biology, Hiram College

2011 to present ...... Graduate Research Associate, Department

of Horticulture and Crop Science, The Ohio

State University

Publications

Michelle Jones and Nichole Edelman. 2013. How to prevent ethylene-related losses during the postproduction care and handling of crops. Greenhouse Management.

Fields of Study

Major Field: Horticulture and Crop Science

Table of Contents

Abstract ...... ii

Acknowledgments...... iv

Vita… ...... v

List of Tables ...... ix

List of Figures ...... x

Chapter 1: Introduction ...... 1

History of Ethylene ...... 1

Ethylene Synthesis ...... 3

Ethylene Signaling and Perception ...... 4

Plant Responses to Ethylene ...... 7

Importance of Ethylene in the Horticulture Industry ...... 11

Ethylene Inhibitors ...... 13

Chapter 2: Comparative Evaluation of Seedling Hypocotyl Elongation and Mature Plant

Screens for Determining Ethylene Sensitivity in Eighteen Bedding Plant Species ...... 19

Abstract ...... 19 vi

Introduction ...... 20

Materials and Methods ...... 23

Seedling Hypocotyl Elongation Screen ...... 23

Plant Culture for Mature Plant Screen ...... 24

Mature Plant Ethylene Treatment ...... 25

Statistical Analysis ...... 26

Results ...... 26

Ethylene Sensitivity of Seedlings ...... 26

Ethylene Sensitivity of Mature Plants ...... 28

Classification of Seedling and Mature Plant Ethylene Sensitivity ...... 30

Discussion ...... 31

Chapter 3: Evaluating Ethylene Sensitivity within the Family Solanaceae at Different

Developmental Stages ...... 62

Abstract ...... 62

Introduction ...... 63

Materials and Methods ...... 66

Plant Materials ...... 66

vii

Seedling Hypocotyl Elongation Screen ...... 67

Plant Culture for Mature Plant Screen ...... 67

Mature Plant Ethylene Sensitivity Screen ...... 68

Evaluation of Tomatoes at Two Developmental Stages ...... 70

Statistical Analysis ...... 70

Results ...... 70

Ethylene Sensitivity within the Solanaceae Family ...... 71

Evaluation of Tomatoes at Two Developmental Stages ...... 70

Discussion ...... 77

References ...... 98

Appendix A: Hypocotyl Elongation in Wild Type and Mutant Arabidopsis Thaliana

Seedlings ...... 109

Appendix B: Hypocotyl Elongation in Wild Type and Transgenic Ethylene Insensitive

Petunia × Hybrida ...... 112

Appendix C: Illustration of how leaf angles were taken in Chapter 3 ...... 115

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List of Tables

Table 2.1. Ethylene classifications from seedling hypocotyl elongation and mature plant screens ...... 37

Table 3.1. Ethylene sensitivity ratings for 41 plant accessions at the seedling and mature plant stages...... 84

List of Figures

Figure 2.1. Evaluation of the hypocotyl elongation screen in 18 bedding plant species .. 39

Figure 2.2. Relative hypocotyl lengths of seedlings germinated on 100 μM ACC ...... 43

Figure 2.3. Flower quality of mature plants immediately after treatment (0 d) with ethylene ...... 44

Figure 2.4. Leaf quality of mature plants immediately after treatment (0 d) with ethylene.

...... 47

Figure 2.5. Visual observations of mature plants after treatment with ethylene gas ...... 49

Figure 2.6. Close up of flower symptoms after treatment with 10 µL·L-1 ethylene for 24 h

...... 50

Figure 2.7. Close up of vegetative symptoms after treatment with 10 µL·L-1 ethylene for

24 h...... 51

Figure 2.8. Flower quality of mature plants 1 d after treatment with ethylene...... 52

Figure 2.9. Leaf quality of mature plants 1 d after treatment with ethylene ...... 55

Figure 2.10. Flower quality of mature plants 2 d after treatment with ethylene ...... 57

Figure 2.11. Leaf quality of mature plants 2 d after treatment with ethylene ...... 60

Figure 3.1. Seedling hypocotyl elongation screen...... 88

Figure 3.2. Flower abscission in mature plants...... 89

Figure 3.3. Flower senescence in mature plants ...... 90

Figure 3.4. Leaf epinasty in mature plants ...... 92

Figure 3.5. Visual observations of leaf epinasty in mature Solanaceous plants after treatment with ethylene gas ...... 93

Figure 3.6. Comparison of seedling and mature plant responses to ethylene for 10

Solanaceous plant accessions ...... 94

Figure 3.7. Effect of mature plant age on ethylene induced leaf epinasty in three

Solanaceous accessions ...... 96

Figure A.1. Evaluation of the hypocotyl elongation screen in wild type and mutant

Arabidopsis thaliana seedlings ...... 109

Figure B.1. Evaluation of the hypocotyl elongation screen in wild type and transgenic ethylene insensitive Petunia × hybrida ...... 112

Figure C.1. Illustrations of how leaf angles were measured in Chapter 3 ...... 115

Chapter 1: Introduction

Ethylene, C2H4, is a plant hormone. Plant hormones are chemicals produced by plants that influence growth and development at low concentrations. Plant hormones are involved in the regulation of everything from seed germination and cell growth, to fruit ripening and the induction of flowering in Angiosperms. Ethylene is unique among other hormones because it is a clear and odorless gas. It is produced by plants during stress, and it is used in the horticulture industry for such things as the induction of fruit ripening. A great deal of research has been done over the years to better understand this hormone and its effects on plants. Products have been developed to inhibit ethylene effects on fruits, crop plants, and ornamentals in both the horticultural and agricultural industries (Taiz and

Zeiger, 2002).

History of Ethylene

The morphological changes that occur to seedlings exposed to ethylene were first reported in the early 20th century (Neljubow, 1901; Knight, 1910). Neljubow observed that dark grown pea seedlings grew horizontally when they were exposed to air containing illuminating gas, while normal vertical growth occurred in the absence of

1 illuminating gas. Ethylene was determined to be the active component of illuminating gas that was responsible for the abnormal seedling growth. This was determined by testing the effect of the individual constituents of illuminating gas including sulfur dioxide, carbon disulfide, benzol, xylol, naphthalene, and ethylene (Neljubow, 1901). Ethylene was the constituent that induced the triple response alone. Symptoms occurring in response to ethylene included an exaggerated apical hook (horizontal growth), thickened hypocotyl, and reduced stem elongation length (Denny, 1924). These responses, collectively termed the triple response, were later used as an ethylene bioassay.

Additional research was conducted by Doubt (1917), who studied the effects of illuminating gas on mature plants. Symptoms of leaf senescence, cell proliferation at the abscission zone, rigor (i.e. cell rigidity, non-responsive to stimulus), petiole epinasty, and root tubercles (root nodules) were reported.

In 1935, ethylene was identified as a natural product of ripening fruit (Gane, 1935), and it was determined to be a plant hormone (Crocker et al., 1935). The physiological responses to ethylene observed by Crocker were similar to those recorded by Doubt. In addition to fruit ripening, Crocker (1935) also observed an inhibition of root elongation and secondary root formations, making the roots appear stunted and hairy. Key research performed in the 1960s and 1970s by Shang Fa Yang and others elucidated the ethylene biosynthesis pathway, termed the Yang cycle. Specific ethylene receptors and binding sites within the pathway were identified by utilizing the triple response as a screen for ethylene mutants in Arabidopsis thaliana accessions (Chang et al., 1993, Lanahan et al.,

1994). Sisler and Blankenship (1996) used this information to develop an ethylene 2 inhibitor, 1-methylcyclopropene (1-MCP), which is used to minimize the deleterious effects of ethylene on mature plants during shipping or on cut flowers to extend shelf life.

Ethylene Synthesis

Ethylene biosynthesis starts with the amino acid methionine, which is converted to S- adenosyl-L-methionine (SAM) by SAM synthetase. SAM is then converted to 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase, which is considered the rate limiting step of ethylene synthesis. Lastly, ACC is converted to ethylene, CO2, and cyanide (HCN) by the enzyme ACC oxidase in the presence of oxygen. An ACC synthase mRNA was first cloned in tomato by using partially purified ACC synthase to produce an antiserum (Sato et al., 1989; Van der Stracten et al., 1989). The antiserum was purified to remove antibodies that do not specifically bind to ACC synthase. The cDNA clones were identified using immunoblotting analysis. ACC synthase cDNA clones were finally expressed and then recovered in E.coli and yeast (Sato et al., 1989). Since then,

ACC synthase has been cloned in many other plants (McManus, 2012).

Ethylene induces the synthesis of both ACC synthase and ACC oxidase. This process is known as autocatalysis (Burg et al., 1967; Yang et al., 1979; Chaves et al., 2006).

Autocatalysis occurs when a catalyst (in this case ethylene) is also one of the products of the reaction. During ripening, an autocatalytic ethylene burst coincides with and accelerates the ripening process in climacteric (i.e. ethylene responsive) fruit, such as tomato and pear (Barry et al., 2000; McMurchie et al., 1972; Blankenship and

Richardson, 1985; Knee, 1987). This autocatalytic burst of ethylene can also be detected

3 as the initial step in flower senescence that accelerates petal wilting (Yang, 1985). As an alternative to blocking the perception of ethylene by using 1-MCP, ACC synthase and

ACC oxidase can be chemically inhibited to prevent ethylene production. Specifically,

ACC synthase is inhibited by aminooxyacetic acid (AOA) or aminoethoxyvinylglycine

(AVG). ACC oxidase is inhibited under anaerobic conditions and by cobalt chloride

(Taiz and Zeiger, 2002).

Ethylene Signaling and Perception

The triple response has been used as a genetic screen for the identification of ethylene mutants, which has lead to an increased understanding of ethylene signaling and perception. The triple response screen consists of germinating seedlings in the presence of ethylene and comparing their morphology with seedlings grown in the absence of ethylene. Seedlings with functional mutations in enzymes involved in the ethylene pathway respond differently to ethylene than wild type seedlings. Screens utilizing the triple response looking for ethylene sensitivity mutants led to the discovery of ethylene receptors and response regulators in Arabidopsis thaliana and Solanum lycopersicum

(tomato). The etr1 (ethylene response 1) mutant was discovered in arabidopsis because it did not display the triple response in the presence of ethylene (Bleecker et al., 1988).

Etr1-1 has a mutation in the ethylene receptor that inhibits ethylene binding, but still allows for negative regulation of the response pathway. This mutation conferred ethylene insensitivity in arabidopsis seedlings (Chang et al., 1993). In contrast, the ctr1 mutant exhibits the triple response even in the absence of ethylene, which revealed the role of

CTR1 as a negative regulator of ethylene in the response pathway (Kieber et al., 1993).

The ein2 mutant exhibits no ethylene response at either the seedling or adult stage in the presence of ethylene, and has also been found in genetic screens for jasmonic acid and abscisic acid resistance (Alonso et al., 1999; Beaudoin et al., 2000). We performed a preliminary experiment using the seedling hypocotyl elongation screen on Arabidopsis thaliana wild type, and ctr1 and ein 2 mutants (Appendix A). When the ctr1 mutant seedlings were compared to wild type Arabidopsis thaliana seedlings, the relative hypocotyl lengths at each concentration were approximately 40% of the wild type control

(0 µM ACC). In contrast the nearly insensitive ein2 mutant seedlings were typically the same length as the wild type control regardless of the presence of ACC. Identified in tomato, an unusual mutant hls1 (hookless) exhibited no apical curvature or “hook” when exposed to ethylene, although symptoms of shortened root length, and thickened hypocotyl were still observed (Lehman et al., 1996). The nr (never ripe) mutant, also in tomato, was discovered to be insensitive to ethylene because of a mutation in the NR ethylene receptor, similar to the etr1 mutant in arabidopsis (Wilkinson et al., 1995;

Leclercq et al., 2002; Klee, 2004).

The discovery of double or triple mutants has also helped to further elucidate the relationship between different genes in this pathway. Phenotypic traits observed in etr1/ ctr1 double mutants mirror the ctr1 mutant. This shows that ctr1 is epistatic to etr1, because it suppresses the effects of the etr1 mutation (Avery and Wasserman, 1992; Taiz and Zeiger, 2002). These and other mutants have revealed a greater understanding of genes involved in the ethylene response pathway. The discovery of insensitive mutants 5 has led to the development of plants with reduced ethylene sensitivity resulting in fruit with longer shelf lives and ornamental plants with longer lasting flowers (Reid and Jiang,

2012).

There are two subfamilies of ethylene receptors, subfamily 1 consists of ETR1 (ethylene resistant 1), and ERS1 (ethylene response sensor 1), the second consists of ERS2, EIN4

(ethylene insensitive 4), and ETR2. These receptors are similar to the two-component histidine kinases found in bacteria (Kieber et al., 1993). Copper ions are required as a cofactor in the binding of ethylene to these receptors. In contrast, silver ions will inhibit ethylene binding. Unbound receptors negatively regulate the ethylene signal transduction pathway. Ethylene works through a negative regulatory response, when the receptors are bound to ethylene they can no longer suppress the ethylene response in plants (Hua and

Meyerowitz, 1998).

CTR1 (constitutive triple response 1) controls kinase activity and interacts with ETR1.

When CTR1 is bound to the ETR1 protein complex it impedes the binding of ethylene to

ETR1, inhibiting the signal for the ethylene response in plants (Nehring and Ecker, 2010).

When ethylene is bound to the receptor it inactivates CTR1, allowing EIN2 to positively regulate the pathway and initiate the ethylene response by sending signals downstream to

EIN3 and other transcription factors that are in the nucleus. EIN3/ EIL1/ EIL2 (EIN3-like genes) bind to ERF1, also a positive regulator, allowing it and other EREBPs (ethylene response element binding proteins) to interact with the GCC box, a genetic sequence found in target promoter genes that is essential to their expression, and results in the

6 promotion of ethylene responses (Wang et al., 2002; Nehring and Ecker, 2010). EREBPs can both activate and repress other transcription factors involved in the ethylene signal pathway.

In Tomato, LeCTR1 was found to be similar to the CTR1 gene found in Arabidopsis. The expression of LeCTR1 in Arabidopsis complemented the ctr1mutant, restoring the wild type phenotype. Through further experiments, six receptors were identified in tomato that were similar to the receptors found in Arabidopsis (Barry et al., 2000; Alexander and

Grierson, 2002).

Plant Responses to Ethylene

When seedlings are exposed to ACC or ethylene they exhibit an exaggerated apical hook, thickened hypocotyl, reduced hypocotyl length, and reduced root hair formation. In more mature plants, it can cause root hair formation; fruit ripening; flower or bud abscission or senescence (wilt); leaf abscission or senescence (chlorosis); and petiole or leaf epinasty.

Other symptoms observed in response to ethylene include inhibition of seed germination, increased auxin transport, inhibition of shoot growth, inhibition of stomatal closing, and the promotion of lateral cell expansion. These symptoms vary depending on ethylene concentration, exposure time, and the sensitivity of the specific plant being exposed

(Abeles et al., 1992; Taiz and Zeiger, 2002).

Symptoms such as leaf or flower abscission, fruit ripening, and root hair formation are irreversible, whereas petiole or leaf epinasty are reversible with time. Within a few hours

7 of treatment with ethylene, severe epinasty of the petioles can be observed in tomatoes and recovery usually takes 2 to 3 days (Abeles et al., 1992). Leaf epinasty is caused by cell expansion on the top side of the petiole, which involves auxin. Epi tomato mutants exhibit epinastic effects due to increasing ethylene levels in the tissues and over expression of ethylene signaling (Barry et al., 2001). Lanahan et al. (1994) compared the ethylene sensitivity of the never ripe (nr) tomato mutant, which produces fruit with decreased ethylene sensitivity and a longer shelf life, to wild type ‘Pearson’ tomatoes at the seedling and mature plant stages. Nr seedlings were classified as ethylene insensitive at the seedling stage because they did not exhibit any symptoms of the triple response when grown on media containing ACC. In contrast, the wild type seedlings exhibited a significant reduction in hypocotyl length. At the mature stage, when plants were infiltrated with ACC, epinasty was only observed in wild type plants, not the nr mutants.

Clark et al. (2001) also utilized the triple response of dark grown Pelargonium × hortorum (geranium) seedlings exposed to ACC (evaluating reduction in hypocotyl elongation) as a screen to determine ethylene sensitivity at the seedling stage. Ethylene inhibits the elongation of seedlings in the dark by inducing the accumulation of the ERF1 gene transcripts (Zhong et al., 2012). Germinating geranium seedlings in the dark is critical for using the triple response screen because in light grown seedlings, ethylene has been shown to induce hypocotyl elongation (Zhong et al., 2012). The severity of ethylene sensitivity was based on hypocotyl measurements. Seedlings with greater hypocotyl elongation were said to be less sensitive to ethylene than seedlings with less elongation.

Mature geranium plant evaluations were conducted on the same cultivars by evaluating 8 petal abscission in response to treatment with exogenous ethylene. Ethylene sensitivity classifications were assigned based on the percent hypocotyl length of seedlings germinated on ACC as compared to control seedlings (no ACC), and the percent of petal abscission in response to ethylene treatment for mature plants. All geranium cultivars tested were determined to have high ethylene sensitivity at both developmental stages.

Additionally, at the seedling stage, the cultivars that required higher ACC concentrations to induce a reduction in hypocotyl length (‘Multibloom Lavender’, ‘Ringo 2000 Salmon’, and ‘Elite White’) also abscised fewer petals at the mature stage in response to ethylene treatment. Thus there was a strong correlation between the seedling and mature plant ethylene screens.

The majority of ethylene sensitivity evaluations have been performed at the mature stage.

These evaluations have provided information on phenotypic responses and the symptom severity in different plants. Woltering (1987) published a protocol for evaluating ethylene sensitivity of mature plants. This protocol evaluated the sensitivity of 52 mature plant species after 24 and 72 hours of treatment with ethylene. Sensitivity classifications were assigned based on the duration of ethylene treatment before an initial response was observed and the severity of that response. These criteria were used to assign sensitivity ratings based on a 0-8 scale. A rating of 0 was assigned to plants that did not respond to treatment with ethylene after 72 hours and a rating of 1-4 was assigned to plants exhibiting a response after 72 hours. For plants exhibiting a response after only 24 hours a rating of 5-8 was assigned. Species were divided into two groups, foliage plants for which symptoms of leaf chlorosis, and leaf epinasty were evaluated, and flowering plants 9 for which bud blasting, flower senescence, leaf chlorosis, leaf epinasty, fruit ripening, and microbial attack were evaluated. Specific symptoms for 52 species representing multiple families were determined, and comparable sensitivity classifications were cataloged based on response severity and exposure time required before symptoms were visible. It was also noted that leaf chlorosis was not a common symptom observed for the majority of the plants evaluated, and often it was not seen until after 72 hours of exposure time. In contrast, it was observed that in 65% of the species evaluated flower abscission and flower senescence were exhibited after only 24 hours of treatment with 15 µL·L-1

(ppm). Van Doorn (2001, 2002) also published studies evaluating the ethylene sensitivity of several mature plant species while only looking at floral symptoms of abscission and senescence. For these species, ethylene sensitivity was based on the duration of exposure before an initial response was observed. Symptom severity was not a factor in classifying sensitivity for these evaluations. For these species only floral symptoms were evaluated, any vegetative symptoms that may have been exhibited in response to ethylene were not included. In the majority of the families that were evaluated, the sensitivity of individual species within a family did not vary much (insensitive to low sensitivity, moderate to high sensitivity, or high to very high sensitivity) with the exception of Amarylidaceae.

Sensitivity for this family ranged from no response (0) to highly responsive (3). Studies such as these including multiple members of a family have been used to evaluate the overall sensitivity of plant families.

When evaluating the ethylene sensitivity within plant families, not all families have been found to have the same level of sensitivity between species or cultivars (Serek and Reid, 10

2000; Macnish et al., 2010). Macnish et al. (2010) evaluated cut flower ethylene sensitivity difference between 38 Rosa hybrida Hybrid Tea cultivars. Significant differences in vase life, petal senescence, petal abscission or leaf abscission were seen among cultivars exposed to ethylene. In 11 cultivars, a significant reduction in flower opening and vase life was exhibited after ethylene treatment, while no change was observed in the cultivars ‘Black Magic’, ‘Cherry Love’, ‘Cool Water’, ‘Esperance’, and

‘Ravel’. Another study was performed by Serek and Reid (2000) evaluating the ethylene sensitivity of 10 cultivars of cut Kalanchoë blossfeldiana flowers. Only one cultivar

(‘Alexandra’) exhibited significant flower senescence after treatment with 0.1 µL·L-1

(ppm) ethylene. For two cultivars (‘Simone’, and ‘Nadia’), 1 µL·L-1 of ethylene was sufficient to induce a response, while in other cultivars (‘Caroline’, ‘Jacqueline’, and

‘Pale Jacqueline’), 10 µL·L-1 ethylene induced a response. One cultivar, ‘Debbie,’ exhibited no response after 24 hours of exposure to 10 µL·L-1 ethylene. These studies have shown that sensitivity differences at the species or cultivar level exist in some families.

Importance of Ethylene in the Horticulture Industry

Ethylene can play both a positive and negative role in the horticulture industry. During production, ethylene based plant growth regulators (PGRs) such as Florel or ethephon can be applied to increase lateral branching, or delay flowering in plants. This provides growers with the ability to grow uniform plants by controlling branching and the approximate time that the plants will flower. The active ingredient in Florel is ethephon, a

11 liquid form of ethylene that can be applied directly to the plant as a spray (Styer, 2002,

Starman et al., 2004). Ethylene is also used to induce ripening in fruit (Abeles et al.,

1992; Taiz and Zeiger, 2002). The negative effects of ethylene can occur during production, packaging, shipping, and retailing. During production, sources of ethylene can include any equipment such as leaf blowers or carts that use combustion engines, malfunctioning heaters which can produce ethylene as a byproduct, and leaky gas lines.

Senescing plant tissue (dead flowers or leaves), wounded plants, and ripening fruit can also be a source of ethylene (Jones and Ling, 2012; Jones and Edelman, 2013). This can be especially damaging during the winter months when there is less ventilation and ethylene gas can accumulate. During packaging, plants can also be wounded during sleeving, which induces the production of ethylene before plants are shipped (Jones and

Ling, 2012). While plants are being transported, they can be physically damaged and are deprived of both light and water for extended periods of time. This stress leads to the accumulation of ethylene in closed boxes or sealed trucks, which can reduce the marketability of the plants. In a retail environment, whole plants or cut flowers may be exposed to ethylene from drought stress, exposure to ripening fruit (in the grocery store), or from other senescing flowers. To negate the effects of ethylene on plants during packaging, shipping, and retailing, ethylene inhibitors are often used (Jones and Edelman,

2013).

Ethylene Inhibitors

Various methods have been developed to inhibit ethylene responses for delayed flower senescence or extended fruit life. Transgenic carnations have been developed with antisense ACC oxidase RNA that delay flower senescence and exhibit longer vase life

(Savin et al., 2005). Transgenic Arabidopsis thaliana and Petunia × hybrida with a mutated etr1-1 receptor are considered to be ethylene insensitive (Chang et al., 1993;

Wilkinson et al., 1997). These plants do not exhibit the triple response as seedlings and have delayed flower senescence as mature plants (Bleecker et al., 1988; Langston et al.,

2004; Chapin and Jones, 2009; Wang et al., 2013). In preliminary experiments, the response to ethylene by wild type petunias and etr1-1 mutants was tested using the seedling hypocotyl elongation screen (Appendix B). When compared to wild type control seedlings (0 µM ACC) etr1-1 mutant seedlings did not show a significant reduction in hypocotyl length at any concentration of ACC. These transgenic petunias have reduced adventitious root formation and lower germination rates, which require treatment with gibberellic acid (GA3) to induce germination (Wilkinson et al., 1997; Clark et al., 1999;

Langston et al., 2004; Wang et al., 2013). In fruit, breeding programs have developed genetically engineered tomatoes and other fruits with delayed ripening. Transgenic antisense ACO oxidase Cucumus melo Chartenais melons exhibit significantly reduced ethylene production, delayed ripening, and abscission. Application of external ethylene can be used to induce abscission of fruit from the vine and ripening (Ayub et al., 1996).

In transgenic tomato, the introduction of a bacterial enzyme, ACC deaminase reduced ethylene production by degrading ACC (Klee et al., 1991). 13

For flowers and fruit varieties that have not been genetically engineered, commercial inhibitors such as silverthiosulfate (STS), 1-methylcyclopropene (1-MCP), 2,5- norbornadiene (NBD), and aminoethyoxyvinylglycine (AVG) are used to block the ethylene response when plants are stressed (such as during shipping) or when exposed to ethylene (Abeles et al., 1992; Taiz and Zeiger, 2002). AVG is commonly used as an ethylene inhibitor to delay fruit ripening (Unrath et al., 2009). Before the development of

1-MCP, NBD was commonly used. NBD is known as a competitive inhibitor, which means that NBD inhibits ethylene binding by competing with the hormone for the same binding sites (Sisler and Pian, 1973). NBD was shown to inhibit ethylene binding in

Dianthus caryophyllus L. ‘White Sim’ cut carnation flowers (Sisler et al., 1986). 1-MCP and STS have also been shown to protect plants from exposure to ethylene (Serek and

Sisler, 2001). STS appears to be effective for a longer time period because the silver ions stay in the tissues of the plant longer, binding to new sites and continuing to block the ethylene response. 1-MCP molecules bind permanently to the ethylene receptors that are already present, so when new binding sites develop they are not blocked by free 1-MCP molecules. This leaves the plant once again vulnerable to the effects of ethylene

(Cameron and Reid, 2001). This is illustrated in Pelargonium peltatum (geranium) that were treated with 1-MCP. After treatment with 1-MCP, plants were exposed to ethylene and no detrimental effects were observed. Sensitivity to ethylene in these plants increased with time as new binding sites developed (Cameron and Reid, 2001).

Several studies have evaluated the efficacy and length of effectiveness of 1-MCP on various horticultural and agricultural crops. An experiment was conducted to analyze the 14 effects of pretreatment with 1-MCP on two cultivars of roses, R. hybrida Kordana

'Vanilla' and R. hybrida Parade 'Bronze' (Müller et al., 2000). Both cultivars were treated with 1-MCP and then placed in the dark under simulated transport conditions of continuous shaking for 4 days. The flowers of untreated 'Vanilla' plants did not exhibit a significant increase in ethylene production after 4 days of transport, but the ethylene production increased in 'Bronze' flowers. When both plants were pretreated with 1-MCP before the dark transport, these plants exhibited improved display life equal to those of the controls (not put under transport conditions). In contrast, when 1-MCP was applied after transport, no difference in shelf life was observed when compared to untreated plants. This showed that 1-MCP needed to be applied before exposure to any stress.

When six geranium cultivars were pretreated with 0.01 µL·L-1 1-MCP for 1 hour before exposure to exogenous ethylene, 1-MCP was effective in reducing petal abscission for all cultivars except ‘Fox’ as compared to control plants (no 1-MCP). When plants were exposed to exogenous ethylene without pretreatment with 1-MCP, ‘Fox’ was determined to be the most sensitive of the 6 cultivars, abscising the most petals. Pretreatment with 1

µL·L-1 1-MCP for 6 or more hours was required before this cultivar exhibited reduced petal abscission equivalent to control plants (Jones et al., 2001)

A study involving both 1-MCP and STS was done on cut flowers of Gentiana scabra

'Shinbisei', G. scabra 'Yuki-hotaru', G. scabra x G. triflora 'Aoikaze', and G. triflora

'Koharu' (Shimizu-Yumoto and Ichimura, 2012). Testing was done on plants that were pollinated after harvest, to study the capacity of 1-MCP to inhibit flower senescence.

Ethylene regulates pollination induced flower senescence in many plants (Burg and

Dijkman, 1967; Stead and Moore, 1979; Whitehead et al., 1984; Halevy et al., 1984;

Nichols, 1971; Goto et al., 1999; Shimizu-Yumoto and Ichimura, 2012). Cut Gentiana flowers were exposed to either STS or 1-MCP for 24 hours. In this experiment, when plants (including both leaves and flowers) were treated with 1-MCP, flower wilting and senescence were delayed in varying levels for all cultivars tested. They proposed that different levels of effectiveness between cultivars may be due to different rates of synthesis of new ethylene receptors, since 1-MCP binds irreversibly to the initial receptors present at exposure (Serek et al., 1995; Sisler et al., 1996; Shimizu-Yumoto and

Ichimura, 2012). A study by Goto et al. (1999) also utilized STS as an inhibitor of ethylene when examining the role of ethylene on senescence of Torenia fournieri flowers. After treatment with 0.02 mM STS only pollinated flowers exhibited extended longevity. In contrast, after treatment with 2 mM both pollinated and stigma-wound induced flowers (which induce ethylene production) exhibited a life span equal to that of controls (no ethylene exposure) before finally senescing. Although the length of effectiveness may be shorter than STS, 1-MCP has the advantage of being environmentally safer with lower animal toxicity, and the same efficacy in lower concentrations (Reid and Staby, 2008). 1-MCP was shown effective on both cut flowers such as carnations (Sisler and Serek, 1996) and on potted plants (Serek and Sisler, 2001).

The container in which the 1-MCP gas is applied can also determine whether or not the plants potentially absorb enough gas to be protected from ethylene. An experiment was conducted to evaluate the absorption of 1-MCP by materials used in packaging plants or 16 fruits during transport. The materials tested included plastic, oak wood, and cardboard.

Dry and wet samples of each media were used in testing. Samples were tested in tightly sealed jars and exposed to 1-MCP over a 24 hour exposure time. After 24 hours, 82% of the initial gas concentration remained in jars containing dry cardboard, while only 1.9% remained in jars with wet cardboard. When apples were exposed in a jar by themselves, only 50% of the initial gas remained after 13 hours, while when apples and cardboard were exposed in the same jars only 10% remained (Vallejo and Beaudry, 2006). This shows the potential absorptive capacity of different packaging materials, which could limit the amount of 1-MCP that is available for uptake by fruits and potted plants. More research needs to be done to address this problem.

In the agricultural industry, Invinsa TM (a product containing 1-MCP as the active ingredient) is currently being used on various crops to protect against ethylene induced damage including flower or kernel abortion, reduced photosynthesis, and drought stress

(The Dow Company, Invinsa TM ). Ethylene has been shown to influence the opening and closing of stomata in response to water stress (Pallas and Kays, 1982; Levitt et al., 1987;

Madhavan et al., 1983; Merritt et al., 2001; Archarya and Assmann, 2009; Wilkinson and

Hartung, 2009; The Dow Company Invinsa informational site). As Invinsa is currently being used to treat against drought stress in agricultural crops, it follows that1-MCP may also help improve resistance to drought stress in horticultural plants as well. However, more research needs to be done to test this idea.

For the postharvest industry, it is especially important to understand the effects of ethylene on crop plants. Symptoms such as flower abscission or leaf chlorosis can make sensitive plants unmarketable after exposure to ethylene produced in response to stress.

Sensitive plants must be treated with ethylene inhibitors such as 1-MCP before the stress of long distance shipping. This is why it is so important to screen mature plants to determine the sensitivity and symptoms exhibited in response to ethylene. This provides growers with information about which plants to treat with 1-MCP before shipping and what new plant introductions are less ethylene sensitive than others. For breeders it provides information on less sensitive varieties for breeding programs, and on sensitive varieties for use in ethylene experiments. A consistent screen for evaluating ethylene sensitivity at the mature plant level can provide sensitivity information on individual plants that is also comparable to other known varieties.

Chapter 2: Comparative Evaluation of Seedling Hypocotyl Elongation and Mature Plant

Assays for Determining Ethylene Sensitivity in Eighteen Bedding Plant Species.

Abstract

Ethylene gas can cause extensive damage to bedding plants during production, shipping, and retailing. Seedlings exposed to ethylene exhibit the triple response, which includes an exaggerated apical hook, thickened hypocotyl, and reduced hypocotyl elongation. Our objective was to determine if the hypocotyl elongation component of the seedling triple response could be used to predict the sensitivity of mature plants at the marketable stage.

Eighteen common bedding plants were evaluated. For the seedling hypocotyl elongation screen, seeds were germinated and grown in the dark on filter paper saturated with various concentrations of 1-aminocyclopropane-1-carboxylic acid (ACC; the immediate precursor to ethylene). The relative hypocotyl length at each ACC concentration was compared to untreated control (0 µM) seedlings. Mature plants, with at least four open flowers, were treated with ethylene (0, 0.01, 0.1, 1, or 10 µl.L-1) in the dark for 24 hours.

Phenotypic responses to ethylene, including flower abscission, flower senescence, leaf abscission, leaf chlorosis, and epinasty were rated on a scale of 0 to 5. Fives species 19 exhibited no reduction in hypocotyl elongation when grown on ACC (low sensitivity).

The remaining species were classified as medium or high ethylene sensitivity at the seedling stage. The most common symptoms of ethylene damage observed in mature plants were leaf epinasty, flower abscission, and flower senescence. The severity of these responses was used to identify plants with high, medium, or low sensitivity to ethylene.

For a few species that were equally responsive at both developmental stages, the seedling hypocotyl elongation screen would prove reliable for predicting the ethylene sensitivity of mature plants.

Introduction

Ethylene (C2H4) is a gaseous plant hormone that is produced by plants during normal development and in response to biotic and abiotic stresses (Abeles et al., 1992). Ethylene is involved in seed germination, stem elongation, abscission, epinasty, senescence, and fruit ripening. Plant responses to ethylene depend on the concentration and exposure time

(i.e. dosage), as well as the plant’s sensitivity to ethylene. Ethylene sensitivity varies by species, ranging from nonresponsive to highly sensitive. Ethylene sensitivity can also vary depending on developmental age or organ type (i.e. flowers, leaves, or roots)

(Abeles et al., 1992; Woodson and Lawton, 1988; Woltering, 1987).

Seedlings that are exposed to ethylene exhibit the triple response, which includes an exaggerated apical hook, hypocotyl thickening, and reduced hypocotyl elongation

(Knight et al., 1910). This response has been used in genetic screens to identify ethylene

20 response mutants, including the etr1-1 mutation, which confers ethylene insensitivity in

Arabidopsis thaliana seedlings (Chang et al., 1993). The seedling triple response screen has helped in successfully identifying many genes involved in both ethylene biosynthesis and perception (Guzman and Ecker, 1990). This screen involves growing seedlings in the dark in the presence of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor to ethylene. In the presence of oxygen, ACC is converted to ethylene by constitutive levels of ACC oxidase in the seedlings (Yang and Adams, 1979). These evaluations must be conducted on dark-grown seedlings, because ethylene promotes hypocotyl elongation in the light (Alonso et al., 1999; Ecker, 1995; Smalle et al., 1997;

Zhong et al., 2012).

The effects of ethylene on hypocotyl elongation are concentration dependent, and hypocotyl length decreases (in the dark) with increasing ethylene or ACC concentration until the response is saturated (Bleecker et al., 1988; Goeschl and Kays, 1975; Lanahan et al., 1994). Clark et al. (2001) utilized the hypocotyl elongation response (a component of the seedling triple response) to evaluate the ethylene sensitivity of dark grown geranium

(Pelargonium ×hortorum) seedlings. Seedlings from all six cultivars showed a reduction in hypocotyl length that correlated with ACC concentration and indicated that they were sensitive to ethylene. The average hypocotyl lengths at various ACC concentrations were used to determine differences in ethylene sensitivity among the cultivars. Petal abscission rates following exposure to various concentrations of ethylene were also used to determine the ethylene sensitivity of mature geranium plants. In this experiment, ethylene sensitivity as determined by the hypocotyl elongation screen correlated with sensitivity as 21 determined by petal abscission. These results suggest that a seedling hypocotyl elongation screen could be used to predict ethylene sensitivity in mature plants, but additional experiments need to be conducted to determine if the seedling hypocotyl response accurately and consistently predicts mature plant sensitivity to ethylene in other species.

Exposure to ethylene during greenhouse production, shipping, and retailing can reduce the shelf life and garden performance of ornamental plants. Ethylene-induced petal senescence, flower abscission, epinasty, leaf chlorosis, and leaf abscission make crops unmarketable and increase postproduction shrink (Jones and Edelman, 2013; Jones and

Ling, 2012). Researchers have investigated the ethylene responsiveness of numerous ornamentals, and these studies have identified plants or flowers that are sensitive or relatively insensitive to ethylene. It is difficult to use these reports to classify the ethylene sensitivity of these plants because the assessments are all based on different ethylene concentrations, durations of treatment, and methods for evaluating responses (Alexander and Grierson, 2002; Barry et al., 2001; Dole et al., 2009; Goto et al., 1999; Ichimura and

Suto, 1998; Serek et al., 1995). Woltering (1987) published a standardized protocol for evaluating ethylene sensitivity that was based on treating mature plants from 52 ornamental species with four concentrations of ethylene for two different exposure times.

The severity of the different ethylene responses was used to classify the plants into a sensitivity class from zero to eight.

It is important for floriculture producers, plant breeders, extension professionals, and researchers to know which plants are sensitive to ethylene and to be able to identify differences in ethylene sensitivity between cultivars. This information can be used by producers to identify the best cultivars for long distance transport, and to determine which crops would benefit from treatment with ethylene inhibitors like 1- methylcyclopropene (1-MCP). It would therefore be extremely valuable to develop a rapid and simple screen for evaluating ethylene sensitivity at the seedling stage, rather than treating large mature plants. Breeders could use this high throughput screen to determine the ethylene sensitivity classification for new plant introductions and to identify less sensitive breeding lines.

The objective of this research was to determine if the seedling hypocotyl elongation screen could be used to predict the sensitivity of mature plants at the marketable stage. To determine the utility of this screen, we developed a protocol for evaluating ethylene sensitivity in seedlings germinated and grown in the dark on filter paper saturated with various ACC concentrations. A comparative study was conducted in mature plants using a modified version of the screening protocols described by Woltering (1987).

Materials and Methods

Seedling Hypocotyl Elongation Screen. The effect of ACC on seedling hypocotyl length was evaluated in 18 bedding plant species (Table 1). ACC stock solutions were filter sterilized (Nalgene Filtration Products, Rochester, New York), and serial dilutions were

23 brought to volume with autoclaved ddH2O. Seeds were sown in Petri dishes containing sterile filter paper saturated with 1000 µL of 0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, or 100

µM ACC. Plates were sealed with parafilm and placed in the dark at 23°C for 7-14 d.

There were ten treatments (concentrations) per cultivar, four replicates (plates) sown per concentration of ACC, and six seeds sown per replicate. The experiment was performed twice, each trial represented one block. Seedlings were scanned using WinRhizo (Regent

Instruments Inc, Canada) imaging software and hypocotyl lengths were measured using

ImageJ Software (Abramoff et al., 2004). The hypocotyl length, as a percentage of the control, was calculated for each seedling and averaged for each treatment (length of seedling/average length of control × 100). This is referred to as the relative hypocotyl length. Seedling ethylene sensitivity was defined as high when relative hypocotyl lengths were below 50%; medium at 50-79%; and low at 80- 100% based on cutoffs determined by statistical analysis at 100 μM ACC. These sensitivity ratings are included in Table 2.1.

Plant Culture for Mature Plant Screen. Seeds were sown in 128-count plug trays using

Promix (Premier Tech Horticulture LTD, Canada) and grown under full-spectrum fluorescent lights. Plants were irrigated by hand as needed and fertilized with 100 mg·L-1

N from 15N-2.2P-12.5K Peter’s Excel Cal-Mag (Everis International BV ICL Fertilizers

Co., Dublin, Ohio). Two to three weeks after germination, seedlings were moved to the greenhouse and transplanted into 4.5 in (11.4 cm) diameter plastic pots. Plants were irrigated by hand as needed, alternating between 150 mg·L-1 N from 15N-2.2P-12.5K

Peter’s Excel Cal-Mag and 250 mg·L-1 N from 20N-4.37P-16.6K Peter’s Professional

(Everis International BV ICL Fertilizers Co., Dublin, Ohio). Average greenhouse 24 temperatures were 26/21 ± 4/2 °C day/night with relative humidity of 45.6% ± 18.3%.

Plants were grown under natural irradiance.

Mature Plant Ethylene Treatments. Experiments were conducted from Sept. 2011 to June

2012. Plants were evaluated at horticultural maturity, which has been defined by Watada et al. (1984) as “the stage of development when a plant or plant part possesses the prerequisites for utilization by consumers for a particular purpose”. In this experiment, our mature plants were market ready bedding plants that had four or more open flowers.

There were five ethylene treatments per cultivar, and four replicates per concentration, with each plant acting as a replicate. The experiment was performed twice and each trial represented one block. Plants were placed into 55 gal (208 L) metal chambers and sealed with Plexiglas lids. Ethylene gas was injected into the chambers to obtain the final concentrations of 0, 0.01, 0.1, 1.0 or 10 µL·L-1, and the chambers were covered to keep plants in the dark. The ethylene concentrations inside the chambers were regularly tested with a gas chromatograph and adjusted as needed (Varian 3800; Agilent, Foster City,

California). Mature plants were removed from the chambers after a 24 h treatment.

Visual observations and photos were taken at 0 (immediately after ethylene exposure), 1,

2, 5, and 7 d. Plants were evaluated for symptoms of flower abscission (drop), flower senescence (wilt), leaf abscission, leaf chlorosis (yellowing), and leaf epinasty

(downward curvature). Flower evaluations did not distinguish between open flowers and buds, and both were included in senescence and abscission ratings. The severity of the ethylene damage on each plant was evaluated using a 0-5 rating scale. Ratings were based on the percentage of the plant exhibiting symptoms, with 5 = 0% (no damage), 4 = 25

1-20%, 3 = 21-40%, 2 = 41-60%, 1 = 61-80% and 0 = 81-100%. For vegetative symptoms a rating of 4 indicated that 1-20% of the leaves showed epinasty or that 1-20% of the leaves were yellowing or had abscised. For floral symptoms, a rating of 4 meant that 1-20% of the flowers on that plant had abscised or wilted (senesced). An overall sensitivity rating for the plant was then assigned based on the ethylene damage symptom that received the highest rating after treatment with 10 µL·L-1 ethylene at 0 d (Table 2.1).

Statistical Analysis. The experiments were conducted using a complete block design and data was analyzed in SAS (version 9.3; SAS Institute, Cary, NC). For the seedling screen, percentages obtained from hypocotyl measurements were analyzed using PROC

GLIMMIX (generalized linear mixed models). All means were adjusted for errors in model and presented as least squares means. Proc Univariate was used to determine that visual observation data for mature plants were normally distributed (i.e. that no data transformation was necessary). Data proved to be normally distributed and therefore was analyzed using PROC GLM (generalized linear model). All means were adjusted for errors in model and presented as least squares means. All statistical tests utilized α = 0.05 for significance.

Results

Ethylene Sensitivity of Seedlings. Thirteen species exhibited a decrease in hypocotyl length with increasing ACC concentrations (high and medium sensitivity), while five species showed little change in hypocotyl length when compared to seedlings germinated

26 with no ACC (low sensitivity) (Fig. 2.1). The greatest decreases in hypocotyl length were observed among pansy, ageratum, phlox, tomato, viola, alyssum, snapdragon and nemesia (Fig. 2.1 A). Snapdragon seedlings responded to concentrations as low as 5 µM, while pansy, viola, and nemesia had an ethylene-induced decrease in hypocotyl length at

10 µM ACC. For ageratum, phlox, tomato, and alyssum initial reductions in hypocotyl length were observed at 50 µM ACC. The relative hypocotyl length at 100 µM ACC varied from 47.2% (pansy) to 33.7% (snapdragon) as compared to controls.

French marigold, portulaca, torenia, petunia, and gomphrena had a smaller reduction in hypocotyl length. The relative hypocotyl length at 100 µM ACC varied from 74.2%

(french marigold) to 57.2% (gomphrena) (Fig. 2.1 B). French marigold, petunia, and gomphrena had a decrease in hypocotyl length at 50 µM ACC, while torenia and portulaca did not show a response until 100 µM. No effects of ACC on hypotocyl length were observed for zinnia, dahlia, lobelia, salvia, or angelonia (Fig. 2.1 C).

For the seedling hypocotyl elongation screen we used ten concentrations of ACC to determine the lowest concentration at which a reduction in hypocotyl length could be observed. While differences in ethylene responsiveness among the species were noted at various ACC concentrations, they were most significant at the 100 µM ACC treatment

(Fig. 2.2). The relative hypocotyl length at 100 μM was therefore used to classify seedlings as having high, medium, or low sensitivity to ethylene (Table 2.1).

Ethylene Sensitivity of Mature Plants. Plants were rated on a scale of 0 to 5 for flower abscission (Fig. 2.3 A), flower senescence (Fig. 2.3 B), leaf epinasty, leaf abscission, and leaf chlorosis after treatment with ethylene (Fig. 2.4). Immediately after exposure to exogenous ethylene for 24 h (0 d), flower abscission was observed in 44% of the species that were evaluated (Fig. 2.3 A, 2.5, 2.6). Open flowers were generally more sensitive to ethylene than buds. Abscission rates were highest among open flowers, although all of the plants that abscised flowers also abscised a few buds. The greatest response was seen in salvia and torenia, with 81-100% flower abscission (rating of 0) after treatment with 10

µL·L-1 ethylene. At this same ethylene concentration, nemesia and tomato, showed 61-

80% flower abscission (rating of 1), while alyssum received an average rating of 2.6, snapdragon 3.5, and phlox and dahlia 3 (~21-40% flower abscission). In contrast, angelonia showed much lower abscission (rating 4.5). Torenia abscised flowers at concentrations as low as 0.01 µL·L-1, while flower loss was first observed in nemesia, phlox, salvia, and tomato treated with 0.1 µL·L-1 ethylene. Alyssum, angelonia, dahlia, and snapdragon did not show significant flower abscission until concentrations of ethylene were 1 µL·L-1. Plants were also rated at 1, 2 (Fig 2.8, 2.9, 2.10, 2.11), 5 and 7 d

(data not shown) after ethylene treatment to determine when the most extensive ethylene damage would be observed. Snapdragon continued to abscise flowers, decreasing from a rating of 3.5 at 0 d to 2 at 5 d (data not shown). The oldest fully open flowers abscised first at 0 d, followed by the younger flowers at the top of the inflorescence on subsequent days. For all other species, no significant increase in flower abscission was observed on

1, 2 (Fig. 2.8 A, 2.10 A), 5 or 7 d (data not shown)

Flower senescence was observed in ten of the 18 species after exposure to ethylene (Fig.

2.3 B, 2.5, 2.6). Immediately after the ethylene treatment (0 d), petunia had the most senescing flowers (rating of 0 at 10 µL·L-1) and petal wilting was observed at concentrations as low as 0.01 µL·L-1. Flower senescence was observed in dahlia, portulaca, and lobelia treated with 0.1 µL·L-1 ethylene. Dahlia received a rating of 4 and both lobelia and portulaca received a rating of 2 at 10 µL·L-1. Angelonia, nemesia, pansy, phlox, viola, and zinnia were given a rating of 4 after 24 h (0 d) of treatment with 10

µL·L-1 ethylene due to the presence of a few wilted flowers. Only open flowers senesced, and by day 2 the buds that had not abscised were opening. Vegetative symptoms of ethylene damage included leaf abscission, chlorosis, and epinasty (Fig. 2.4, 2.5, 2.7).

Symptoms of leaf epinasty were observed in ageratum, dahlia, french marigold, tomato, and salvia exhibited leaf epinasty (downward curvature of the leaf petiole at 0 d (Fig. 2.4,

2.5 F, 2.7 B, 2.7 C). The most severe epinastic response was observed in tomato (0 rating) and ageratum (1.5 rating) at 10 µL·L-1. Tomato and ageratum responded to ethylene concentrations as low as 0.1 µL·L-1. Epinasty was observed in french marigold at 1 µL·L-1, and plants received a rating of 3 at 10 µL·L-1. Dahlia responded with leaf epinasty after treatment with 10 µL·L-1 ethylene (3.5 rating). Epinasty in french marigolds was only observed in the uppermost (youngest) leaves (top 30%) (Fig. 2.7 B) and full recovery (back to a rating of 5) was observed within 24 hours (by the 1 d observation; Fig. 2.9). Dahlia and salvia were also fully recovered at 1 d (Fig. 2.9).

Tomato and ageratum had recovered to a rating of 5 (no observable epinastic bending) at

2 d (Fig. 2.11).

The only species to exhibit leaf abscission was portulaca, while the only species to exhibit leaf chlorosis was alyssum. Extensive leaf abscission was observed on 0 d when portulaca were treated with 10 µL·L-1 ethylene (Fig. 2.4, 2.5 B). Plants lost approximately half of their leaves and received an average rating of 2.5. A response was observed at concentrations as low as 0.1 µL·L-1. Portulaca continued to lose leaves at 1 d, and the average rating decreased to 2.0 (Fig. 2.9). The only species to exhibit leaf chlorosis was alyssum, where the lower (oldest) leaves showed yellowing, and an average rating of 3.5 was assigned to plants treated with 10 µL·L-1. We continued to observe symptoms and record ratings through 7 d to determine if there would be any delays in the visual appearance of leaf chlorosis after removal from the ethylene gas. We did not observe any changes in the extent of leaf chlorosis over time (Fig. 2.9, 2.11, data not shown).

The severity of symptoms varied among species (Table 2.1). Gomphrena was the only species that did not exhibit a response to the ethylene treatment. It was classified as no response instead of insensitive because it may respond to higher ethylene dosages (i.e. increased exposure time and/ or increased ethylene concentrations). Ageratum, french marigold, lobelia, pansy, petunia, snapdragon, torenia, viola and zinnia each exhibited only a single symptom (flower abscission, flower senescence, leaf abscission, leaf chlorosis, or leaf epinasty). In contrast, alyssum, angelonia, dahlia, nemesia, phlox, portulaca, salvia and tomato exhibited multiple symptoms when treated with ethylene.

Classification of Seedling and Mature Plant Ethylene Sensitivity. Seedlings and mature plants of the 18 bedding plant species were grouped into one of three ethylene sensitivity

30 classifications (Table 2.1). While most of the species had similar trends in ethylene responsiveness between the seedling stage and the mature stage, there were four exceptions. Lobelia and salvia were classified as low sensitivity at the seedling stage and high at the mature stage, while pansy and viola had low sensitivity at the mature stage and were classified as having high sensitivity at the seedling stage.

Discussion

Our research was conducted to determine whether the seedling hypocotyl elongation screen could be used to predict the ethylene sensitivity of market ready bedding plants at horticultural maturity. The seedling hypocotyl elongation screen was previously used to identify zonal geranium cultivars with reduced sensitivity to ethylene, and these differences in sensitivity were validated in mature plants using a petal abscission assay

(Clark et al., 2001). We found that the seedling screen did not always predict mature plant ethylene sensitivity. The most striking differences between the seedling and mature plant screens were observed in lobelia, salvia, viola, and pansy. The hypocotyl elongation screen indicated that lobelia and salvia seedlings had low sensitivity to ethylene, while mature plant screens indicated that they had high sensitivity due to the high rates of flower senescence and abscission, respectively, that were observed following a 24 h treatment with 10 µL·L-1 ethylene. Both viola and pansy showed the opposite response, with seedlings exhibiting high sensitivity and mature plants receiving a low sensitivity rating due to the ethylene-induced senescence of only a few flowers. There is very little information available on ethylene responses in the genus Viola. The Flower and Plant

Care Guide (Nell and Reid, 2000) indicates that pansies are not sensitive to ethylene, while Rogers (1985) identifies pansy as one of the crops with intermediate susceptibility to damage from ethylene pollution. For these four species in particular, the seedling screen would not provide an adequate prediction of the ethylene sensitivity of mature, market ready plants.

In six of the 18 species, the seedling screen and the mature plant screen resulted in identical classifications. These included ageratum, angelonia, nemesia, tomato, marigold, and zinnia. While it may be possible to consistently predict mature plant sensitivity using the seedling hypocotyl elongation screen in these species, additional cultivars should be evaluated to determine if the response is consistent between cultivars. Ethylene sensitivity differences have been described between cultivars and varieties within a species (Woodson and Lawton, 1988; Sisler et al., 1996; Serek and Reid, 2000; Jones et al., 2001; Serek and Sisler, 2001; Macnish et al., 2010). In carnations, which are generally considered to have high sensitivity to ethylene, breeding programs have selected for reduced ethylene sensitivity to create cut carnation varieties with extended vase life (Onozaki et al., 2001).

In an additional eight species (alyssum, dahlia, gomphrena, petunia, phlox, portulaca, snapdragon, and torenia), classifications differed by one category. In phlox, for example, seedlings were classified as high sensitivity and mature plants were classified as medium sensitivity. While these classifications are only approximate, they are based on responses that were consistently observed among plant replicates and between independent

32 experiments. These classifications may also vary slightly from those reported in the literature due to the specific cultivars that were selected for evaluation (Nell and Reid,

2000). In this evaluation, the seedling response was based on quantitative measurements of hypocotyl length, while mature plant classifications relied on a more subjective visual rating system to evaluate the severity of the response. Therefore it may be possible to improve on the reliability of these classifications by using a more quantitative assessment of mature plant responses. We will address this in future experiments by quantifying the exact percentage of senescing and abscising organs (i.e. flowers and leaves) and the degree of epinastic curvature of leaves (chapter 3).

Contrasting effects of ethylene have been observed between plant species, but also within different plant organs and at different developmental stages (Smalle and Van Der

Straeten, 1997). These responses are influenced by environmental factors including light

(Alonso et al., 1999; Ecker, 1995; Le et al., 2005; Smalle et al., 1997; Zhong et al., 2012).

The responsiveness of flower petals to ethylene is dependent on the physiological age of the tissue. In most species, flower buds are less sensitive to ethylene damage than open flowers, and ethylene sensitivity increases from flower bud to anthesis (i.e. flower opening) (Borochov and Woodson, 1989). In carnation flowers, ethylene sensitivity then decreases from anthesis to senescence (Onozaki et al. 2004). In contrast, ethylene sensitivity increases from anthesis to senescence or abscission in petunia (Whitehead and

Haley, 1989), geranium (Evensen, 1991), portulaca (Ichimura and Suto, 1998), and

Torenia (Goto et al., 1999). The differences observed in our work between seedlings and

33 mature plants can be explained by these developmental differences in ethylene responsiveness.

We used ten concentrations of ACC in our seedling screens to determine which concentrations resulted in a reduction in hypocotyl elongation and to quantify the extent of that reduction compared to untreated control seedlings. We found that the percent hypocotyl reduction at 100 µM as compared to controls (i.e. the relative hypocotyl length) identified the most significant differences and allowed us to classify the species into low, medium and high sensitivity. Angelonia, dahlia, lobelia, salvia and zinnia hypocotyl length (at 100 µM ACC) was not significantly reduced compared to controls (0

µM ACC). Relative hypocotyl length was consistently below 100%, so these species were classified as having low ethylene sensitivity rather than classifying them as insensitive. It is possible that higher concentrations of ACC could be required to inhibit hypocotyl elongation in these species. For most species, using only 0 and 100 µM ACC should be sufficient to quickly screen for seedling ethylene sensitivity. For the purposes of identifying plants that can be damaged by ethylene, and which would benefit from 1-

MCP treatment, this simplified approach and the resulting three sensitivity classifications will be adequate. A more comprehensive dose response analysis may be required to identify more subtle differences between cultivars.

The triple response screen used to identify ethylene response mutants involves germinating seedlings on Murashige and Skoog (MS) media containing ACC (Clark et al., 2001; Guzman and Ecker, 1990). For our hypocotyl elongation screen, we germinated

34 the seedlings on sterile filter paper that was saturated with ACC. This approach saved time and resources because large volumes of MS media did not have to be prepared, and seeds did not require sterilization. Time saving modifications, like these, make our seedling hypocotyl elongation screen a much more efficient high throughput screen for ethylene sensitivity.

Many excellent experiments have been conducted to evaluate the ethylene sensitivity of foliage plants, flowering plants, and cut flowers (Dole et al., 2009; Macnish et al., 2011;

Woltering, 1987; Woltering and Van Doorn, 1988). Woltering (1987) evaluated 52 ornamental pot plants using a protocol that involved treating mature plants with four concentrations of ethylene for two different exposure times. Flower abscission and senescence were observed after a 24 h ethylene treatment (at up to 15 µL·L-1), while most vegetative symptoms were not observed until after 72 h of treatment. Flower abscission was the most common ethylene symptom, occurring in 65% of the species evaluated, and leaf senescence was the least common symptom occurring in less than 4% of the species

(Woltering, 1987). In our study, we observed symptoms of leaf epinasty in five out of the

18 species, whileethylene-induced leaf chlorosis and abscission were observed only in alyssum and portulaca, respectively. We may have seen increased vegetative symptom severity if our 10 µL·L-1 treatment went for 72 hours or longer. We observed flower senescence (50% of species) and flower abscission (44% of species) at a similar frequency in the 18 bedding plants that were selected for evaluation.

Mature plant responses are often similar (in symptoms and extent) among plants within a family (Van Doorn, 2001; Van Doorn, 2002). While the seedling hypocotyl elongation screen cannot be used to predict unknown plant sensitivity without preliminary evaluations of mature plants, it may be possible to use this screen for a high throughput analysis of species and cultivars within plant families where ethylene sensitivity at the seedling stage has been shown to correlate with mature plant sensitivity. The development of a simple and reliable screen for evaluating seedling and mature plant sensitivity will provide breeders, growers, transporters, and retailers with critical information about a plant’s ethylene sensitivity. These screens can be used to evaluate new plant introductions and to identify cultivars that are less sensitive to ethylene and which are better suited for long distance transport. Growers and transporters can also use this information to determine which crops should be treated with ethylene inhibitors like

1-MCP to enhance their postproduction quality. Plants in both the medium and high classifications would benefit from postproduction treatment with 1-MCP to prevent symptoms of ethylene damage.

Table 2.1. Ethylene classifications from seedling hypocotyl elongation and mature plant screens.

Species Seedling Mature plant Mature plant symptoms x

sensitivity z sensitivity y

Ageratum houstonianum 'High Tide Blue' high high leaf epinasty

Angelonia angustifolia ‘Serena Lavender Pink' low low flower abscission, flower

senescence

Nemesia fruticans 'Poetry Blue' high high flower abscission, flower

senescence

Solanum lycopersicum (tomato) 'Tumbler' high high flower abscission, leaf epinasty

Tagetes patula (french marigold) 'Durango Bee' medium medium leaf epinasty

Zinnia marylandica 'Double Zahara Fire' no response low flower senescence

Antirrhinum majus (snapdragon) 'Bedding Rocket Lemon' high medium flower abscission

Dahlia × hybrida 'Figaro White' low medium flower abscission, flower

senescence, leaf epinasty

Gomphrena globosa 'Fireworks' medium no response none

Lobularia maritima (alyssum) 'Snow Crystals' high medium flower abscission, leaf chlorosis

Petunia × hybrida 'Carpet White' medium high flower senescence

Phlox drummondii 'Palona Light Salmon' high medium flower abscission, flower

senescence

Portulaca grandiflora 'Margarita Scarlet' medium high flower senescence, leaf abscission

Torenia fournieri 'Clown Blue' medium high flower abscission

Lobelia erinus 'Regatta Rose' low high flower senescence

Salvia splendens 'Red Hot Sally' low high flower abscission, leaf epinasty

Viola cornuta 'Sorbet XP Orange' high low flower senescence

Viola × witrockiana (pansy) 'Matrix Lemon' high low flower senescence

continued

Table 2.1. continued z Seedling sensitivity was based on the relative hypocotyl length at 100 μM ACC compared to controls (0 μM ACC). The classifications included low (80-100%), medium

(50-79%), and high (below 50%). y Mature plant sensitivity was determined by sensitivity ratings (0-5) taken at 0 d in response to treatment with 10 µL·L-1 exogenous ethylene. The classification low (0-20%; rating 5 and 4), medium (21-40%; rating 3), and high (41-100%; rating 2, 1, and 0) was assigned based on the lowest rating (i.e. the most severe symptom) received for an observed symptom. x Symptoms observed when horticulturally mature plants were treated with ethylene for

24 h.

Figure 2.1. Evaluation of the hypocotyl elongation screen in 18 bedding plant species.

Seeds were sown on filter paper saturated with 1-aminocyclopropane-1-carboxylic acid

(ACC) at 0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, or 100 μM. Seedling hypocotyl length was measured at 7-14 d after germination. Bars represent the relative hypocotyl length as a percent of the control (0 μM ACC) for each concentration. Error bars represent the standard error, and letters above the error bars represent significance between treatments based on separation presented as the least squares means using α = 0.05 for significance.

Plant species were categorized as having high (A), medium (B), or low (C) ethylene sensitivity.

Figure 2.1. continued

A continued

Figure 2.1. continued

B continued

Figure 2.1. continued

Figure 2.2. Relative hypocotyl lengths of seedlings germinated on 100 μM ACC. Bars represent the hypocotyl length at 100 μM ACC as a percentage of the controls (0 μM

ACC). Error bars represent the standard error and letters above the error bars represent significance between species based on the separation presented as the least squares means using α = 0.05 for significance. Species were categorized as having high (white bars), medium (gray bars) or low (black bars) ethylene sensitivity.

Figure 2.3. Flower quality of mature plants immediately after treatment (0 d) with ethylene. Species exhibiting flower abscission (A) and flower senescence (B) after treatment with 0, 0.01, 0.1, 1, or 10 µL·L-1 ethylene for 24 h. Ratings were assigned based on the percentage of flowers on the plant showing symptoms: 5 = 0%, 4 = 1-20%,

3 = 21-40%, 2 = 41-60%, 1 = 61-80%, 0 = 81-100%. Bars represent the average visual rating, error bars represent the standard error, and letters above the error bars show significance based on separation presented as the least squares means using α = 0.05 for significance.

Figure 2.3. continued

A continued

Figure 2.3. continued

B continued

Figure 2.4. Leaf quality of mature plants immediately after treatment (0 d) with ethylene.

Species exhibiting leaf epinasty, leaf abscission, and leaf chlorosis after treatment with 0,

0.01, 0.1, 1, or 10 µL·L-1 ethylene for 24 h. Ratings were assigned based on the percentage of leaves on the plant showing each symptom: 5 = 0%, 4 = 1-20%, 3 = 21-

40%, 2 = 41-60%, 1 = 61-80%, 0 = 81-100%. Bars represent the average visual rating, error bars represent the standard error, and letters above the error bars show significance based on separation presented as the least squares means using α = 0.05 for significance.

Figure 2.4. continued

Figure 2.5. Visual observations of mature plants after treatment with ethylene gas.

Torenia (A), marigold (B), pansy (C), portulaca (D), lobelia (E) and tomato (F) are shown immediately after treatment (0 d) with 0, 0.01, 0.1, 1, or 10 µL·L-1 for 24 h (seen from left to right). 49

Figure 2.6. Close up of flower symptoms after treatment with 10 µL·L-1 ethylene for 24 h.

Photos were taken on 0 d, immediately after removal from ethylene, and show flower abscission in salvia (A) and torenia (B) and flower senescence in portulaca (C) and petunia (D). 50

Figure 2.7. Close up of vegetative symptoms after treatment with 10 µL·L-1 ethylene for

24 h. Photos were taken on 0 d, immediately after removal from the ethylene chamber, and show leaf abscission in portulaca (A) and leaf epinasty in french marigold (B) and tomato (C).

Figure 2.8. Flower quality of mature plants 1 d after treatment with ethylene. Species exhibiting flower abscission (A) and flower senescence (B) after treatment with 0, 0.01,

0.1, 1, or 10 µL·L-1 ethylene for 24 h. Ratings were assigned based on the percentage of flowers on the plant showing symptoms: 5 = 0%, 4 = 1-20%, 3 = 21-40%, 2 = 41-60%, 1

= 61-80%, 0 = 81-100%. Bars represent the average visual rating, error bars represent the standard error, and letters above the error bars show significance based on separation presented as the least squares means using α = 0.05 for significance.

Figure 2.8.

A continued

Figure 2.8. continued

Figure 2.9. Leaf quality of mature plant 1 d after treatment with ethylene. Species exhibiting leaf epinasty, leaf abscission, and leaf chlorosis after treatment with 0, 0.01,

0.1, 1, or 10 µL·L-1 ethylene for 24 h. Ratings were assigned based on the percentage of leaves on the plant showing each symptom: 5 = 0%, 4 = 1-20%, 3 = 21-40%, 2 = 41-

60%, 1 = 61-80%, 0 = 81-100%. Bars represent the average visual rating, error bars represent the standard error, and letters above the error bars show significance based separation presented as the least squares means using α = 0.05 for significance

Figure 2.9. continued

Figure 2.10. Flower quality of mature plants 2 d after treatment with ethylene. Species exhibiting flower abscission (A) and flower senescence (B) after treatment with 0, 0.01,

0.1, 1, or 10 µL·L-1 ethylene for 24 h. Ratings were assigned based on the percentage of flowers on the plant showing symptoms: 5 = 0%, 4 = 1-20%, 3 = 21-40%, 2 = 41-60%, 1

Figure 2.10 continued

A continued

Figure 2.10. continued

B 59

Figure 2.11. Leaf quality of mature plants 2 d after ethylene treatment. Species exhibiting leaf epinasty, leaf abscission, and leaf chlorosis after treatment with 0, 0.01, 0.1, 1, or 10

µL·L-1 ethylene for 24 h. Ratings were assigned based on the percentage of leaves on the plant showing each symptom: 5 = 0%, 4 = 1-20%, 3 = 21-40%, 2 = 41-60%, 1 = 61-80%,

0 = 81-100%. Bars represent the average visual rating, error bars represent the standard error, and letters above the error bars show significance based on separation presented as the least squares means using α = 0.05 for significance.

Figure 2.11

Chapter 3: Evaluating Ethylene Sensitivity within the Family Solanaceae at Different

Developmental Stages.

Abstract

Ethylene (C2H4) is a hormone that is produced by plants during growth and development and in response to environmental stress. Seedlings that are exposed to ethylene have reduced hypocotyl elongation, thickened hypocotyls, and exaggerated apical hooks. This group of symptoms is referred to as the seedling triple response. Ethylene gas causes abscission, accelerated senescence, and epinasty in mature plants. The specific symptom and the severity of the response to ethylene varies between species and even among cultivars. The family Solanaceae, which includes both important crop and ornamental species, is generally considered to have high sensitivity to ethylene, but a detailed evaluation of members of this family has not been conducted at both the seedling and mature plant stages. Our objectives were to evaluate ethylene sensitivity between accessions with the family Solanaceae, and to determine whether similar sensitivity is observed at seedling and mature plant stages. Seedlings were germinated on 1- aminocyclopropane-1-carboxylic acid (ACC) and sensitivity was determined based on the reduction in hypocotyl length observed in response to ACC. Mature plants were treated with 10 µL·L-1 ethylene gas for 24 h, and the severity of the response (flower abscission, flower senescence, or leaf epinasty) was used to assign ethylene sensitivity

62 classifications of high, medium, or low. Thirty-five out of 41 plant accessions were classified as medium or high ethylene sensitivity and only six plants exhibited low sensitivity at both the seedling and the mature plant stages. Our results indicated that there was not a consistent correlation between seedling and mature plant sensitivity within the family Solanaceae. Information from this study can be used by producers to identify varieties with reduced ethylene sensitivity for long-distance shipping or to identify varieties that would benefit from treatment with ethylene inhibitors, like 1- methylcyclopropene (1-MCP).

Introduction

Ethylene (C2H4) is a gaseous hormone that is produced by plants during growth and development and in response to environmental stress (Abeles et al., 1992). At the seedling stage, ethylene causes reduced hypocotyl elongation, increased hypocotyl thickening, and an exaggerated apical hook. Collectively these symptoms are known as the triple response (Knight, 1910). Exposure to ethylene at plant maturity can lead to flower, bud or leaf abscission, flower senescence, leaf chlorosis (yellowing), or epinasty

(downward curvature of the leaf or petiole) (Abeles, 1992). The quality of ornamental plants is reduced by exposure to ethylene during production, shipping, and retailing

(Jones and Edelman, 2013; Jones and Ling, 2012). Ethylene also controls fruit ripening in many species, and the ripening process is accompanied by a burst of ethylene production

(Burg and Burg, 1965; Alexander and Grierson, 2002). Decreasing ethylene production

63 and inhibiting ethylene perception increases the postharvest shelf life of ethylene sensitive fruits, cut flowers, and potted plants.

Most evaluations of ethylene sensitivity have been performed on whole plants or on detached organs (i.e. fruits or cut flowers) (Woltering, 1987; Woltering and Van Doorn,

1988; Serek et al., 1994; Serek et al., 1995; Goto et al., 1999; Van Doorn, 2001; Van

Doorn, 2002; Archambault, 2006; Dole et al., 2009). A few studies have used the seedling triple response to evaluate differences in ethylene sensitivity between plants

(Lanahan et al., 1994; Clark, 2001; Chapter 2). The seedling triple response screen has been extremely successful at identifying ethylene biosynthesis and signaling mutants in tomato (Solanum lycopersicum) and arabidopsis (Arabidopsis thaliana) (Alexander and

Grierson, 2002; Binder et al., 2004). In dark-grown seedlings, hypocotyl length decreases at increasing concentrations of ethylene or 1-aminocyclopropane-1-carboxylic acid

(ACC), the immediate precursor to ethylene, and this component of the triple response provides an easy screen for evaluating ethylene sensitivity. In 2001, Clark et al. used the seedling hypocotyl elongation screen to compare ethylene sensitivity between geranium

(Pelargonium ×hortorum) cultivars. Petal abscission rates after treatment with ethylene gas were used to evaluate ethylene sensitivity in mature plants. In these six geranium cultivars, ethylene sensitivity at the seedling stage correlated with ethylene sensitivity in mature plants. The ethylene sensitivity of the never ripe (nr) tomato mutant and wild type

‘Pearson’ tomatoes was compared at the seedling and mature plant stages (Lanahan et al.,

1994). Nr seedlings were classified as ethylene insensitive, because they did not exhibit any symptoms of the triple response when grown on media containing ACC. In contrast, 64 the wild type seedlings exhibited a significant reduction in hypocotyl length. When mature plants were infiltrated with ACC, epinasty was observed in only the wild type plants, not the nr mutants.

Solanaceae is a relatively large and very diverse family that includes many plants that are highly sensitive to ethylene. The family contains approximately 90 genera consisting of

3000-4000 species, which include edible, ornamental, and medicinal plants (Bombarely et al., 2011). Solanum melongena (eggplant), Capsicum annum (pepper), Solanum tuberosum (potato), Solanum lycopersicum (tomato), and Physalis ixocarpa (tomatillo) are important food crops, while Calibrachoa × hybrida, Petunia × hybrida, and the genus Nicotiana include popular ornamentals. Nicotiana, petunia, and tomato are also widely used as model experimental plants for studying development and plant-pathogen interactions (Wing et al., 1994; Gerats and Vandenbussche, 2005; Goodin et al., 2008).

While studies evaluating ethylene sensitivity have been published for multiple

Solanaceous plants at the seedling or mature plant stages (Lanahan et al., 1994; Van

Doorn, 2001; Van Doorn, 2002; Chaabouni et al., 2009, Chapter 2), a more comprehensive screen comparing sensitivity between genera, species, and cultivars within this family has not been conducted.

We have evaluated ethylene sensitivity in 41 different plant accessions. This study included two objectives: 1) to identify ethylene sensitivity differences (levels of sensitivity and symptoms) between accessions within the Solanaceae family; and 2) to identify ethylene sensitivity differences at different developmental stages (seedling,

65 juvenile, and mature plants). The information obtained from this study can be used by horticultural producers to select less sensitive varieties for long-distance transport and to identify plants that would benefit from treatment with ethylene inhibitors, like 1- methylcyclopropene (1-MCP). This information will also be useful for plant breeders and scientists who want to identify breeding lines with lower ethylene sensitivity.

Materials and Methods

Plant Materials. Forty Solanaceous plants, representing six genera, were selected for evaluation (Table 3.1). Plants included wild species and cultivated varieties. Zinnia marylandica ‘Double Zahara Fire’ (not a member of the family Solanaceae) was included as a negative control, because this plant was known to have little to no response to ACC at the seedling stage or to exogenous ethylene at the mature plant stage (Chapter 2). For simplicity, all of the plants in these experiments will be referred to as accessions.

Solanum pimpinellifolium LA1589 seeds were provided by Dr. Esther Van der Knaap

(The Ohio State University). Solanum habrochaites LA0407, Solanum lycopersicum var.

Cerasiformae accession PI114490, Solanum lycopersicum ‘Selección Mallorquín’,

Solanum lycopersicum ‘FL7600’, Solanum lycopersicum ‘OH8819’, Solanum lycopersicum ‘OH9242’, Solanum lycopersicum ‘OH981067’, Solanum pennellii

LA0716, and Solanum pimpinellifolium PI128216 seeds were provided by Dr. David

Francis (The Ohio State University). Nicotiana tabacum ‘Xanthi’ seeds were provided by

Dr. Feng Qu (The Ohio State University). Calibrachoa × hybrida cuttings were provided

66 by Green Circle Growers (Oberlin, Ohio), and all other seeds were obtained from the Ball

Horticultural Company (West Chicago, Illinois).

Seedling Hypocotyl Elongation Screen. The effect of 1-aminocyclopropane-1-carboxylic acid (ACC) on seedling hypocotyl length was evaluated for the species listed in Table 3.1 except calibrachoa, which were available only as vegetative cuttings, and tomato

‘Seleccion Mallorquin’ and ‘OH9492’, which had very inconsistent germination rates.

Seeds were sown onto sterile filter paper saturated with 1000 µL of 0 (control) or 100 µM

ACC in Petri dishes. This concentration was chosen based on previous experiments

(Chapter 2). The Petri dishes were sealed with parafilm and incubated in the dark at 23°C for 7-10 d. There were two treatments (ACC concentrations) consisting of four replicates

(plates), with six seeds sown per replicate. The experiment was performed twice, and each trial represented one block. Seedlings were scanned using WinRhizo (Regent

Instruments Inc, Canada) imaging software, and hypocotyl lengths were measured using

ImageJ Software (Abramoff et al., 2004). The hypocotyl length, as a percentage of the control, was calculated for each seedling and averaged for each treatment (seedling length/mean control length × 100). Seedling ethylene sensitivity classifications were determined based on the percentage of the hypocotyl length at100 µM ACC as compared to control seedlings (0 µM ACC): low= 80-100%; medium= 50-79%; and high= below

50%. This value is referred to as the relative hypocotyl length.

Plant Culture for Mature Plant Screen. Seeds were sown in 128-count plug trays using

Promix (Premier Tech Horticulture LTD, Canada), grown under full-spectrum

67 fluorescent lights, and fertigated as needed with 100 mg·L-1 N from 15N-2.2P-12.5K

Peter’s Excel Cal-Mag (Everis International BV ICL Fertilizers Co., Dublin, Ohio).

Seedlings were transported to the greenhouse approximately 3 weeks after germination.

Plants were grown under natural irradiance with supplemental lighting provided by high pressure sodium and metal halide lamps (GLX/GLS e-systems GROW lights;

PARsource, Petaluma, CA). The average photosynthetic photon flux was ≈ 225 μmol· m-

2· s-1 from 0600-1800 h. Average greenhouse temperatures were 22/20 ± 2/1 °C day/night with 49.2% ± 18.1% relative humidity. After 1 week in the greenhouse, plants were transplanted into 4.5-inch-diameter plastic pots and irrigated by hand as needed alternating between 150 mg·L-1 N from 15N-2.2P-12.5K Peter’s Excel Cal-Mag and 250 mg·L-1 N from 20N-4.37P-16.6K Peter’s Professional (Everis International BV ICL

Fertilizers Co., Dublin, Ohio).

Mature Plant Ethylene Sensitivity Screen. Mature plant evaluations were conducted from

July 2012 to June 2013 on all of the accessions in Table 3.1. Plants were placed into 208

L (55 gallon) metal treatment chambers and sealed with Plexiglass lids. Ethylene gas was injected into the chambers to a final concentration of 10 µL·L-1 (ppm). Control chambers contained air (0 µL·L-1ethylene). The ethylene concentrations inside the chambers were confirmed regularly using a gas chromatograph with a flame ionization detector (Varian

3800; Agilent, Foster City, California). The chambers were covered to exclude light, and plants were treated for 24 h. Mature plants were treated at the flowering stage (more than five flowers), except for select commercial and wild tomato accessions that flowered later and were evaluated only for leaf epinasty. Each evaluation included two ethylene 68 treatments and four replicates (plants) per treatment. The experiment was performed twice, and each experiment represented one block. The majority of the plants were evaluated for symptoms of flower abscission (drop), flower senescence (wilt), leaf abscission, leaf chlorosis (yellowing), and leaf epinasty (downward curvature) immediately after removal from the chambers (0 d) and at 1, 2, 5 and 7 d after treatment.

Leaf abscission and leaf yellowing were not observed on any of the plant accessions, so detailed methods are not provided for these evaluations. Ten tomato accessions were evaluated only for leaf epinasty (Table 3.1). The total number of flowers (including open flowers and buds) on each plant was counted before treatment (ethylene-treated and controls) so that the percent flower abscission and flower senescence could be calculated at each time point (0, 1, and 2 d). Initial leaf angles were measured for each plant before treatment and epinasty was reported as the change in leaf angle at each time point compared to the initial leaf angle (Appendix C). Leaf angles were obtained for the bottom six leaves per plant by measuring the angle between the stem and the top of the petiole.

These six values were used to calculate the average epinastic curvature per plant.

Measurements were taken until leaf angles were within 10° of the initial angle measurements and plants were considered to be recovered. Sensitivity classifications were assigned for each symptom based on the percent response (flower abscission or flower senescence at 2 d) or the change in the leaf angle (leaf epinasty at 0 d) after exposure to ethylene (Chapter 2). Flower responses at 2 d after treatment were used for assigning sensitivity classifications because some plants had a delayed response once removed from the chambers, and flower wilting and abscission rates were greatest at 2 d.

In contrast, the epinastic response was most severe immediately after the ethylene treatment (0 d) and many plants had recovered by 1 or 2 d. Ethylene sensitivity classifications for each symptom were as follows: low= 0-20% floral abscission or senescence, 0-20° change from initial leaf angle; medium= 21-40% floral abscission or senescence, 21-40° change from initial leaf angle; or high= 41-100% floral abscission or senescence, greater than 40° change from initial leaf angle. An overall sensitivity rating for the mature plant was then assigned to each accession based on the highest rated symptom (Table 3.1).

Evaluation of Tomatoes at Two Developmental Stages. In a separate experiment,

Solanum lycopersicum (tomato) ‘Brandywine Pink Potato Leaf’, ‘Super Sweet 100’, and

‘Tumbler’ plants were evaluated at two developmental stages (juvenile and mature).

Stage 1 juvenile plants were evaluated approximately 4-5 weeks after sowing, had 6-8 leaves, and were not yet flowering. Stage 2 mature plants were evaluated 8-9 weeks after sowing, had more than 8 leaves and were flowering. Data was collected only for leaf epinasty. Plants were treated with ethylene and evaluated as described above.

Statistical analysis. All of the experiments were conducted using a randomized complete block design and data was analyzed in SAS (version 9.3; SAS Institute, Cary, NC). For the seedling screen, percentages obtained from hypocotyl measurements were analyzed using PROC GLM (generalized linear model). Significance was determined based on a mean separation using the least significant difference (LSD) test. Data collected from mature plant responses to ethylene were analyzed by PROC GLIMMIX (generalized

70 linear mixed models). Significant means were separated using LSD. All statistical tests utilized α = 0.05 for significance.

Results

Ethylene Sensitivity within the Solanaceae Family. Screening was conducted on seedlings and mature plants to evaluate ethylene sensitivity differences between accessions within the family Solanaceae. At the seedling stage, most accessions (32 out of 36) were classified as having high or medium ethylene sensitivity (Fig. 3.1). Only four accessions had relative hypocotyl lengths of 80- 100% when seedlings were grown on 100 µM ACC and compared to the hypocotyl lengths of control seedlings grown on 0 µM ACC. These accessions were classified as low sensitivity at the seedling stage and included Zinnia marylandica ‘Double Zahara Fire’, Solanum melongena (eggplant) 'Black Beauty’,

Nicotiana paniculata, and Capsicum annum (pepper) 'Habanero Big Sun'. Symptoms observed in mature plants after treatment with 10 µL·L-1 ethylene for 24 h included flower abscission, flower senescence, and leaf epinasty. Zinnia ‘Double Zahara Fire’ and

Solanum pennellii LA0716 were the only accessions that did not exhibit any symptoms of ethylene damage, and they were categorized as having low sensitivity (Fig. 3.2, 3.3, 3.4,

3.6, Table 3.1). Zinnia (not a member of the family Solanaceae) was included as a negative control because this accession was known to have little to no response to ACC at the seedling stage or exogenous ethylene at the mature plant stage (Chapter 2). For each accession, control plants were treated with 0 µL·L-1 ethylene for 24 h. No significant

71 flower abscission, flower senescence, or leaf epinasty was observed in any of the control plants (data not shown).

Three accessions of Calibrachoa × hybrida were chosen for evaluation. No data on the ethylene sensitivity of seedlings was obtained because commercial calibrachoa are propagated vegetatively. After exposure to 10 µL·L-1 ethylene, flower senescence was the main symptom of ethylene damage observed in all three cultivars (Fig. 3.3). Only a few flowers abscissed (Fig. 3.2; 5-11% of the total flowers), and no leaf damage was noted

(e.g. leaf chlorosis, abscission or epinasty) (Fig. 3.4). The extent of flower senescence between the cultivars at 0 d (immediately after removal from ethylene) varied from 10-

40%. The percentage of senescing flowers increased over time (Fig. 3.3). Calibrachoa

‘Noa Sunrise’ had the fewest senescing flowers at 2 d (37%) and received an overall sensitivity rating of medium, while ‘Terra Cotta’ and ‘Dark Blue’ were classified as high ethylene sensitivity due to flower senescence rates of 55% and 93% (2 d), respectively

(Fig. 3.3B, Table 3.1.)

At the seedling stage, three accessions of Petunia × hybrida (‘Madness Red’, ‘Dreams

Salmon’, and ‘Daddy Orchid’) were classified as having high sensitivity to ethylene, with relative hypocotyl lengths less than 50% of the controls (0 µM ACC). Petunia ‘Mitchell

Diploid’, ‘Easy Wave Blue’, and ‘Carpet White’ were classified as medium sensitivity with average relative hypocotyl lengths which varied from 54- 59% (Fig. 3.1). As with calibrachoa, flower senescence was the main symptom of ethylene damage observed in mature plants (Fig. 3.3). ‘Madness Red’ and ‘Carpet White’ were the most sensitive,

72 senescing 47% and 41% of flowers at 2 d (Fig. 3.3 B). These two accessions were assigned an overall sensitivity rating of high. ‘Dreams Salmon’, ‘Easy Wave Blue’, and

‘Mitchell Diploid’ senesced 4- 11% of flowers at 0 d (Fig. 3.3 A) and 24- 28% of the flowers were senescing by 2 d (Fig. 3.3 B), resulting in a medium ethylene sensitivity rating. ‘Daddy Orchid’ was the only petunia to receive a low sensitivity rating at the mature plant stage. This accession had very little flower senescence at 0 d (4%) and only

19% of the flowers senesced by 2 d.

Eight accessions of Nicotiana, that included seven different species, were selected for evaluation. Seedling sensitivity ranged from low (N. paniculata) to high (N. mutabilis

‘Marshmallow’) with relative hypocotyl lengths which varied from 82% to 52% (Fig.

3.1). The majority of the nicotiana accessions (6 out of 8) were classified as having medium ethylene sensitivity at the seedling stage (Table 3.1). Mature plant accessions were classified as having medium (N. benthamiana, N. alata ‘Lime Green’, N. silvestris

‘Only the Lonely’, and N. paniculata) and high (N. mutabilis ‘Marshmallow’, N. alata

‘Starmaker Appleblossum’, N. suaveolens, and N. tabacum ‘Xanthi’) ethylene sensitivity

(Table 3.1). Flower abscission was the main symptom of ethylene damage in mature

Nicotiana plants, and very little flower senescence (below 20%) was observed. While N. mutabilis ‘Marshmallow’ had no initial response to ethylene (0%; Fig. 3.2 A), 43% of the flowers had abscised by 2 d (Fig. 3.2 B). N. tabacum ‘Xanthi’ exhibited the greatest flower abscission at 0 d (48%), with no additional abscission observed on subsequent days (Fig. 3.2).

Seedling ethylene sensitivity of both Solanum melongena (eggplant) accessions varied from low (‘Black Beauty’, 96% relative hypocotyl length) to medium (‘Classic’, 69% relative hypocotyl length) (Fig. 3.1, Table 3.1). At the mature plant stage, both accessions were assigned an overall sensitivity rating of high based on flower abscission rates which increased from 0-2 d (Fig. 3.2, Table 3.1). ‘Classic’ exhibited the most severe response, increasing from 37% to 81% flower abscission by 2 d, while ‘Black Beauty’ only increased from 40% to 53%. ‘Black Beauty’ and ‘Classic’ senesced 11% and 20% of their flowers at 2 d (Fig. 33 B). Both accessions received a low sensitivity rating for this symptom (Table 3.1). No leaf epinasty was observed in ethylene treated eggplants.

Three accessions of Capsicum annum (pepper) including ‘Bell Yellow’, ‘Bell California

Wonder 300’, and ‘Habanero Big Sun’ were evaluated and seedling ethylene sensitivity ranged from low to medium (Table 3.1). ‘Bell Yellow’ and ’Bell California Wonder 300’ both exhibited a medium response, with average relative hypocotyl lengths of 69% and

61%, respectively. No reduction in hypocotyl length was observed for ‘Habanero Big

Sun’ (low sensitivity) when compared to the controls (0 µM ACC) (Fig. 3.1). Symptoms observed in mature plants after ethylene treatment included flower abscission, flower senescence, and leaf epinasty. ‘Habanero Big Sun’ also had the lowest ethylene sensitivity at the mature plant stage, with flower abscission and senescence rates under

20% at 2 d (low rating) and only a 30° change from initial leaf angle at 0 d (medium rating) (Fig. 3.2, 3.3, 3.4, 3.5 A). ‘Bell Yellow’ abscised 30% of its flowers at 0 d and

48% of the flowers by 2 d (Fig. 3.2). ‘Bell California Wonder 300’ abscised 44% of the flowers at 0 d with no significant change over the next 2 d (Fig. 3.2), and 27% of the 74 flowers were senesced by 2 d (medium rating, Fig. 3.3). Both ‘Bell Yellow’ and ‘Bell

California Wonder 300’ exhibited little change in leaf angle (9-14°) at 0 d (low rating)

(Fig. 3.4 A, 3.5 B). In general, symptoms of epinasty were greatest on 0 d, immediately upon removal from ethylene, and the extent of epinastic curvature in the leaves decreased from 0 to 2 d. ‘Habanero Big Sun’ and ‘Bell California Wonder 300’ recovered to within

10° of their original leaf angles within 1 d (Fig. 3.4 B), while ‘Bell Yellow’ was fully recovered by 2 d (Fig. 3.4 C, 3.5 B).

For Physalis ixocarpa (tomatillo) ‘Grande Rio Verde’, relative seedling hypocotyl length was 69%, resulting in a seedling ethylene sensitivity classification of medium (Fig. 3.1,

Table 3.1). ‘Grande Rio Verde’ was given a mature plant rating of high sensitivity based on leaf epinasty. Tomatillo showed one of the most severe examples of epinastic curvature among the Solanaceae, with a 69° change in epinastic curvature at 0 d (Fig. 3.4,

3.5 C). This accession also showed some ethylene induced flower damage, receiving a low rating for flower senescence (5%) and a medium rating for abscission (28%) (Fig. 3.2

B, Table 3.1).

Seven commercial cultivars of Solanum lycopersicum (tomato) were evaluated in a screen of seedling and mature plant ethylene sensitivity. At the seedling stage, ‘Tumbler’ was classified as having medium sensitivity, while all other accessions were classified as having high ethylene sensitivity (Table 3.1). The average relative hypocotyl lengths ranged from 55% (‘Tumbler’) to 26% (‘Better Boy’) (Fig. 3.1). While ‘Tumbler’ was the least sensitive at the seedling stage (Fig. 3.1), ‘Tumbler’ and ‘Brandywine Pink Potato

Leaf’ both exhibited the greatest epinastic curvature at the mature plant stage (68°) (Fig.

3.4 A, 3.5 E). ‘Micro Tom’ had the least severe leaf epinasty (28°). The epinastic curvature of the other cultivars varied from 51- 65° immediately after ethylene treatment

(0 d) (Fig. 3.4 A). Rates of flower abscission at 0 d ranged from 7-25 % and increased to

14-73% at 2 d (Fig. 3.2 A, 3.2 B). The only accession to exhibit flower senescence at 0 d was ‘Tumbler’. Leaf epinasty was the most severe symptom of ethylene damage and determined the overall sensitivity rating for all mature tomato plants (Table 3.1, Fig. 3.5

D, 3.5 E), except ‘Micro Tom’ (Table 3.1). ‘Micro Tom’ received a high sensitivity rating based on flower abscission (73%), while epinasty (28°) resulted in only a medium sensitivity rating (Fig. 3.2 B. 3.4 A).

A further evaluation of tomato included additional accessions of S. lycopersicum and wild relatives S. habrochaites, S. pennellii, S. pimpinellifolium, and S.lycopersicum var.

Cerasiformae. Ethylene sensitivity was evaluated using both the seedling and mature plant screens, but epinasty was the only symptom that was quantified in the mature plants. At the seedling stage, relative hypocotyl length varied from 33%- 21% and seedling sensitivity was classified as high for all accessions (Fig. 3.6 A). Epinastic curvature varied from 25- 54° at 0 d for nine out of 10 cultivars (classified as medium to high for the symptom of epinasty) (Table 3.1, Fig. 3.6 B). S. pennellii LA0716 was classified as high sensitivity at the seedling stage, and low sensitivity at the mature stage with no epinasty observed after treatment with ethylene (0 d) (Fig. 3.6).

Evaluation of Tomatoes at Two Developmental Stages. The symptom of epinasty was further evaluated for three selected cultivars of tomato (‘Tumbler’, ‘Brandywine Pink

Potato Leaf’, and ‘Super Sweet 100’) at two stages of development (stage 1 and stage 2)

(Fig. 3.7). For all three accessions, the greatest epinastic curvature was observed in the juvenile plants (stage 1, at 4-5 weeks). Stage 1 ‘Brandywine Pink Potato Leaf’ plants exhibited epinastic curvature of 82°, while stage 2 mature plants had epinastic curvature of only 56°. The most extreme effect of plant age on ethylene induced epinasty was observed in ‘Tumbler’, which had a difference in epinastic curvature of over 60° in the leaf angles observed in stage 1 (102°) and stage 2 (35°) plants (Fig. 3.7). At stage

1‘Tumbler’ would be classified as high sensitivity while the stage 2 plants would receive a medium sensitivity classification.

Discussion

Our research was conducted to identify differences in ethylene sensitivity between members of the family Solanaceae, and to classify ethylene damage symptoms and sensitivity differences at multiple developmental stages. When seedling and mature plant screens were compared, sensitivity classifications matched in 37% of the plants. For most

Solanaceae accessions, sensitivity at both developmental stages was medium or high, with only six accessions classified as low at either the seedling or mature plant stage.

Members of the family Solanaceae are generally reported as ethylene sensitive, and the family contains many model species used to study ethylene’s role in flower senescence, fruit ripening, seedling development, and pathogen responses (Wing et al., 1994; Gerats

77 and Vandenbussche, 2005; Goodin et al., 2008). Seedling and mature plant ethylene sensitivities were opposite (low vs high or high vs low) for only 3 accessions. Solanum melongena (eggplant) ‘Black Beauty’ was classified as low sensitivity at the seedling stage and high at the mature stage. Petunia × hybrida ‘Daddy Orchid’, and Solanum pennellii LA0716 were classified as high ethylene sensitivity at the seedling stage and low at the mature stage. These results correspond with our previous study (Ch. 2) in which 4 out of 18 of the plants that were evaluated had the opossitve sensitivity classifications when seedling and mature plant sensitivity were compared. While Clark et al., (2001) demonstrated that the seedling hypocotyl elongation screen was a good predictor of mature plant sensitivity in geranium, we found that Viola cornuta ‘Sorbet XP

Orange’, Viola × wittrockiana (pansy) ‘Matrix Lemon’, Lobelia erinus ‘Regatta Rose’, and Salvia splendens ‘Red Hot Sally’ had opposite sensitivity responses in mature plants and seedlings (Ch. 2).

Developmental stage (juvenile vs mature) also influenced the severity of the ethylene response in tomato. For two out of the three cultivars (‘Tumbler’ and ‘Super Sweet 100’),

4-5 week old juvenile plants (stage 1) had epinastic curvature that corresponded to a high ethylene sensitivity response, but 8-9 week old mature plants (stage 2) had only a medium response. Contrasting ethylene responses have been reported in different plant organs and at different developmental stages in multiple plant species (Smalle and Van

Der Straeten, 1997; Suttle and Hultstrand, 1991). These studies illustrate why it is important to use plants at equivalent developmental stages for ethylene treatments and to accurately report the age of these plants when publishing the results from ethylene 78 sensitivity screens. We recommend that ethylene sensitivity evaluations in plants that exhibit epinasty should be conducted in juvenile plants unless there is a corresponding interest in evaluating floral symptoms.

The main noticeable effects of ethylene treatment on the above ground portion of the plant include both floral and vegetative symptoms. Damage to the foliage includes leaf abscission, leaf senescence, and epinasty, and floral damage includes flower senescence and the abscission of petals or whole flowers. After exposure to 10 µL·L-1 ethylene for 24 h, epinasty was the only symptom of vegetative damage that was observed in the 40

Solanaceous accessions that we evaluated. Epinasty is a symptom that has commonly been reported in tomato, and is often observed in response to flooding or other abiotic stresses that induced ethylene production in the shoots (English et al., 1995). We consistently observed ethylene-induced epinasty in the genus Capsicum and in all of the

Solanum except eggplant (S. melongena). Epinasty was not observed among any accessions of petunia, nicotiana, or calibrachoa. The severity of the epinastic response varied between species and even between accessions within a species like S. lycopersicum.

Tomato is often suggested as an indicator plant to detect ethylene contamination from faulty heaters in commercial greenhouses due to its very visual epinastic curvature when exposed to low concentrations of ethylene. Even among tomatoes, we observed differences in the severity of the epinasty response. In our evaluation, ‘Tumbler’,

‘Brandywine Pink Potato Leaf’, and ‘Better Boy’ exhibited the greatest leaf epinasty, and

79 they would therefore serve as the best ethylene indicator plants in a greenhouse. We would suggest using juvenile tomato plants and replacing them before they flower.

Differences in ethylene sensitivity were also observed among ten accessions of wild tomato relatives including S. habrochaites, S. pennellii, S. pimpinellifolium, and S. lycopersicum var. Cerasiformae. Accessions ranged from medium to high ethylene sensitivity at both the seedling and mature plant stage except S. pennellii, which was classified as having high ethylene sensitivity at the seedling stage and low sensitivity at the mature plant stage. This species exhibited no change in leaf angle at the mature stage after ethylene treatment for 24 h. When S. pennellii LA0716 was treated with 10 µL·L-1 ethylene for 72 hours, minor epinasty of the petiole was observed (data not shown), illustrating that this species is not ethylene insensitive. The lack of epinastic response may be due in part to the structure of the petioles and stem of LA0716, which are more rigid than typical tomato leaves. S. lycopersicum ‘OH981067’ and ‘Seleccion

Mallorquin’ have mutations in the Nac Nor gene, which confers low ethylene sensitivity and extended shelf life in fruit (Kopeliovitch et al., 1980; Dias et al., 2003). In other studies, fruit from these plants were shown to be less ethylene sensitive, but leaf evaluations were not performed. During our evaluations, it was shown that this mutation did not result in reduced ethylene sensitivity in other floral or vegetative tissues. These accessions were classified as having medium and high ethylene sensitivity respectively, based on the epinastic response of the petioles after exposure to ethylene. Since our mature plant screen did not include evaluations of fruit, this screen would not be

80 reflective of fruit shelf life, and illustrates the importance of tissue specific ethylene testing when evaluating ethylene sensitivity.

With the exception of epinastic curvature of leaf petioles, vegetative tissues tend to be less sensitive to ethylene than floral tissues, and higher doses are required to induce leaf senescence and abscission (Woltering, 1987; Serek and Trolle, 2000; Kadner and Druege,

2004). In a comprehensive study of ethylene sensitivity conducted by Woltering (1987),

50% of foliage and 30% of flowering plants abscised some leaves. This response was mainly seen when plants were exposed to higher concentrations of ethylene for longer periods of time (i.e. higher doses). In his study, Capsicum annuum (pepper) exhibited leaf abscission, but only after treatment with 15 µL·L-1ethylene for 72 hours. The younger leaves were more sensitive to the ethylene treatment and abscised before the older leaves.

Ethylene generally promotes the abscission of the oldest leaves on the plant first, but this young leaf abscission response has also been reported in cotton (Morgan and Durham,

1973; Suttle and Hultstrand, 1991). Solanum pseudocapsicum (Jerusalem Cherry) vegetation was more sensitive to ethylene and abscised leaves after only 24 hours of exposure to ethylene (Woltering, 1987). We may have observed leaf abscission in some of our accession if we had included additional treatments that went longer than 24 h, but these responses would still have resulted in a low ethylene sensitivity classification.

Many of the Solanaceous accessions that we evaluated are sold as ornamental or vegetable bedding plants. Bedding plant crops can be exposed to damaging concentrations of ethylene during production, shipping, or retailing, and this causes

81 flower loss due to premature senescence or abscission (Jones and Edelman, 2013; Jones and Ling, 2012). It was for this reason that we included the quantification of ethylene- induced flower damage symptoms for the majority of our accessions. In our study, pepper, tomato, and tomatillo exhibited both floral and vegetative symptoms of ethylene damage, while calibrachoa, eggplant, nicotiana, and petunia exhibited only floral symptoms. The predominant floral symptom in the family Solanaceae was flower abscission, which was observed in almost all of the Solanum, Capsicum, and Nicotiana accessions. Calibrachoa and petunia were characterized by higher rates of flower senescence, but many of these flowers also abscised once they were fully wilted.

Ethylene responses in flowers are fairly consistent within families, and most can be characterized as having either ethylene-induced flower abscission or senescence or ethylene-insensitive senescence (van Doorn, 2001). Other evaluations of flowers within the family Solanaceae have identified abscission as the primary response, but often flowers wilt slightly first and then abscise. This is in contrast to some flowers that abscise only fully turgid corollas, with no prior wilting (van Doorn, 2001; Woltering, 1987;

Woltering and van Doorn, 1988). Nierembergia sp. and Browallia speciosa, which are important ornamentals within the family Solanaceae also exhibit ethylene-induced abscission of flowers and buds (Woltering, 1987; van Doorn, 2001). The severity of ethylene responses in flowers varies between cultivars, as we observed among the petunia and calibrachoa cultivars evaluated in this study. Evaluations of rose, carnation, and kalanchoe have identified varieties that have short shelf lives (or vase lives) due to high sensitivity to ethylene, and those where an extended flower life is correlated with

82 decreased ethylene sensitivity (Woodson and Lawton, 1988; Sisler et al., 1996; Serek and

Reid, 2000; Serek and Sisler, 2001; Macnish et al., 2010).

While previous evaluations indicated that mature plant responses between members of a family are often similar (Van Doorn, 2001; Van Doorn, 2002), this study has illustrated the variety of responses and severity of symptoms observed between plants within the family Solanaceae. Symptoms were more consistent within genera, but the severity of the responses varied between accessions within a species. A mature plant screen including both floral and vegetative observations at a consistent developmental stage is required for determining mature plant sensitivity and evaluating differences in sensitivity at the cultivar or species level. The insight gained from this experiment provides researchers, growers, and breeders with more information on the ethylene sensitivity of Solanaceous plants. While any classifications of this nature can only be approximate, they are based on reliable comparisons between accessions, and the responses were consistently observed within replicates and between independent experiments. Identification of sensitivity differences between accessions within the Solanaceae family can be used to identify less ethylene sensitive cultivars to use for long distance shipping, which breeding lines may have reduced ethylene sensitivity. It is most valuable to know which plants are responsive to ethylene and would benefit from treatment with 1-MCP. Any plants classified as medium or high ethylene sensitivity should be treated with 1-MCP to prevent damage symptoms which may make the plants unmarketable.

Table 3.1. Ethylene sensitivity ratings for 41 plant accessions at the seedling and mature plant stages.

Plant Accessions Ethylene Sensitivity Plant Symptom Rating

Seedlingz Mature Flower Flower Leaf

Planty Abscission Senescence Epinasty

Zinnia marylandica 'Double Zahara low no response no response no response no response

Fire'

Calibrachoa × hybrida 'Dark Blue' NDx high low high no response

Calibrachoa × hybrida 'Noa NDx medium low medium no response

Sunrise'

Calibrachoa × hybrida 'Terra NDx high low high no response

Cotta'

Solanum melongena 'Black Beauty' low high high medium no response

Solanum melongena 'Classic' medium high high low no response

Nicotiana benthamiana medium medium medium low no response

Nicotiana alata 'Starmaker medium high high low no response

Appleblossum'

Nicotiana alata 'Lime Green' medium medium medium low no response

Nicotiana mutabilis 'Marshmallow' high high high low no response

Nicotiana silvestris 'Only The medium medium medium low no response

Lonely'

Nicotiana paniculata low medium medium low no response

Nicotiana suaveolens medium high high no response no response

Nicotiana tabacum ‘Xanthi’ medium high high low no response

continued 84

Table 3.1. continued Capsicum annum 'California medium high high medium low

Wonder 300'

Capsicum annum 'Yellow' medium high high medium low

Capsicum annum 'Habanero Big low medium low low medium

Sun'

Petunia × hybrida 'Carpet White' medium high low high no response

Petunia × hybrida 'Daddy high low low low no response

Orchid'

Petunia × hybrida 'Dreams high medium low medium no response

Salmon'

Petunia × hybrida 'Easy Wave medium medium low medium no response

Blue'

Petunia × hybrida 'Madness high high low high no response

Red'

Petunia × hybrida 'Mitchell medium medium low medium no response

Diploid'

Physalis ixocarpa 'Grande Rio medium high medium low high

Verde'

Solanum lycopersicum high high low low high

'Beefsteak'

Solanum lycopersicum 'Better high high low low high

Boy'

Solanum lycopersicum high high low low high

'Brandywine Pink

Potato Leaf'

Solanum lycopersicum 'Cherokee high high high low high

Purple'

continued 85

Table 3.1. continued

Solanum lycopersicum 'Micro high high high low medium

Tom'

Solanum lycopersicum 'Super high high low low high

Sweet 100'

Solanum lycopersicum 'Tumbler' medium high high low high

Solanum habrochaites LA0407 high medium NDw NDw medium

Solanum lycopersicum var. high medium NDw NDw low

‘Cerasiforme’ PI114490

Solanum lycopersicum ‘Seleccíon NDw high NDw NDw high

Mallorquín’

Solanum lycopersicum ‘FLA7600’ high medium NDw NDw medium

Solanum lycopersicum ‘OH8819’ high high NDw NDw high

Solanum lycopersicum ‘OH9492’ NDw medium NDw NDw medium

Solanum lycopersicum high medium NDw NDw medium

‘OH981067’

Solanum pennellii LA0716 high no response NDw NDw no response

Solanum pimpinellifolium high medium NDw NDw medium

LA1589

Solanum pimpinellifolium high medium NDw NDw medium

PI128216

continued

Table 3.1. continued z Seedling sensitivity ratings were based on percent relative hypocotyl length in response to ACC at 100μM as compared to controls (0μM ) and classified as low (80-100% relative hypocotyl length), medium (50-79% relative hypocotyl length) or high (below

50% relative hypocotyl length) ethylene sensitivity.

y Overall mature plant sensitivity was determined based on the highest sensitivity rating assigned for flower abscission, flower senescence, or leaf epinasty for each plant accession. Flower abscission and flower senescence symptom sensitivity ratings were determined by the percent response observed 2 d after treatment with 10 µL·L-1 ethylene and classified as low (0-20%), medium (21-40%), or high (above 40%) sensitivity to ethylene. Leaf epinasty sensitivity ratings were determined by the degree of epinastic curvature which is defined as the difference in leaf angle at 0 d when compared to the initial leaf angle measured before treatment with ethylene and classified as low (0-20° change from initial leaf angle), medium (21-40° change from initial leaf angle), and high

(greater than 40° change from initial leaf angle) sensitivity to ethylene. x Not determined. Calibrachoa seedling sensitivity was not determined because seeds were not available. Seedling sensitivity for Solanum lycopersicum ‘Seleccíon Mallorquín’ and ‘OH9495’ were not determined due to inconsistent germination rates. w Floral symptoms were not recorded for these species.

Figure 3.1. Seedling hypocotyl elongation screen. Seedlings were evaluated 7- 10 d after germination. Bars represent the relative hypocotyl length at 100 μM ACC expressed as a percentage of the control treatment (0 μM ACC). Error bars represent the standard error, and letters above error bars represent significance between accessions which are based on least significant means test (α = 0.05). Plant species were classified as having low (black bars), medium (gray bars), or high (white bars) ethylene sensitivity.

Figure 3.2. Flower abscission in mature plants. Rates of flower abscission were determined at 0 d (A) and 2 d (B) after treatment with 10 µL·L-1 ethylene for 24 h. Bars represent the average percentage of abscised flowers for each accession. Error bars represent the standard error, and letters above the error bars represent significance between accessions which are based on least significant means test (α = 0.05). Accessions were classified as having low (black bars), medium (gray bars) or high (white bars) ethylene sensitivity. 89

Figure 3.3. Flower senescence in mature plants. Rates of flower senescence were determined at 0 d (A) and 2 d (B) after treatment with 10 µL·L-1 ethylene for 24 h. Bars represent the average percentage of senesced flowers for each accession. Error bars represent the standard error, and letters above the error bars represent significance between accessions which are based on least significant means test (α = 0.05). Accessions were classified as having low (black bars), medium (gray bars) or high (white bars) ethylene sensitivity. 90

Figure 3.4. Leaf epinasty in mature plants. The severity of leaf epinasty was determined at 0 d (A), 1 d (B), and 2 d (C) after treatment with 10 µL·L-1 ethylene for 24 h. Bars represent the average change in the leaf angle for each accession as compared to initial leaf angles before treatment. Error bars represent the standard error, and letters above the error bars represent significance between accessions which are based on least significant means test (α = 0.05). Accessions were categorized as having low (black bars), medium

(gray bars) or high (white bars) ethylene sensitivity.

Figure 3.4.

Figure 3.5. Visual observations of leaf epinasty in mature Solanaceous plants after treatment with ethylene. (A) C. annum ‘Habanero Big Sun’, (B) C. annum ‘Bell Yellow’,

(C) P. ixocarpa ‘Grande Rio Verde’, (D) S. lycopersicum ‘Cherokee Purple’, and (E) S. lycopersicum ‘Tumbler’ are shown at 0 d after treatment with 0 µL·L-1 ethylene for 24

H, and at 0 d and 2 d after treatment with 10 µL·L-1 ethylene for 24 h (seen from left to right). 93

Figure 3.6. Comparison of seedling and mature plant responses to ethylene for 10

Solanum accessions. (A) Seedlings were evaluated 7 d after germination. Bars represent the average relative hypocotyl length at 100 μM ACC expressed as a percentage of the control treatment (0 μM ACC). Error bars represent the standard error, and letters above the error bars represent significance between accessions. Accessions were categorized as having low (black bars), medium (gray bars) or high (white bars) ethylene sensitivity. (B)

Effects of ethylene on accessions exhibiting leaf epinasty on 0 d after treatment with 10

µL·L-1 ethylene for 24 h. Bars represent the degree change at 0 d from initial leaf angles taken before treatment. Error bars represent the standard error, and letters above the error bars represent significance between accessions which are based on least significant means test (α = 0.05). Accessions were categorized as having low (black bars), medium

(gray bars) or high (white bars) ethylene sensitivity. Hypocotyl length not determined

(ND) for S. lycopersicum ‘Seleccion Mallorquin’ and ‘OH9242’ due to inconsistent germination rates.

Figure 3.6. continued

Figure 3.7. Effect of mature plant age on ethylene induced leaf epinasty in three

Solanaceous accessions. (A) The severity of leaf epinasty was determined at 0 d after treatment with 10 µL·L-1 ethylene for 24 h. Bars represent the average change in the leaf angle for each accession as compared to initial leaf angles before treatment. Error bars represent the standard error, and letters above the error bars represent significance between accessions which are based on least significant means test (α = 0.05). Black bars represent stage 1 plants; grey bars represent stage 2 plants. Stage 1 plants are 4-5 weeks old with 6-8 leaves and no flowers and stage 2 plants are 8-9 weeks old flowering plants with more than 8 leaves. (B) Visual observations of leaf epinasty in stage 1 and stage 2 S. lycopersicum ‘Tumbler’ plants at 0 d after 24 h treatment with 10 µL·L-1 ethylene.

Figure 3.7. continued

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Appendix A: Evaluation of the Hypocotyl Elongation Screen In Wild Type And Mutant

Arabidopsis Thaliana Seedlings.

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Figure A.1. Evaluation of the hypocotyl elongation screen in wild type and mutant

Arabidopsis thaliana seedlings (Dr. Jyan-Chyun Jang, The Ohio State University).Seeds were sown on filter paper saturated with 1-aminocyclopropane-1-carboxylic acid (ACC) at 0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, or 100 μM. Hypocotyl length was measured at 7 d after germination. Bars represent the percent of hypocotyl length as compared to the control (0 μM ACC Arabidopsis thaliana, wild type) for each concentration, error bars represent the standard error, and letters above error bars represent significance between treatments based on least squares means test using α = 0.05 for significance. (A)

Arabidopsis thaliana (wild type), (B) Arabidopsis thaliana ctr1, and (C) Arabidopsis thaliana ein2.

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Figure A.1. continued

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Appendix B: Evaluation of the hypocotyl elongation screen in wild type and transgenic

ethylene insensitive Petunia × hybrida.

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Figure B.1. Evaluation of the hypocotyl elongation screen in wild type and transgenic ethylene insensitive Petunia × hybrida (Wilkinson et al., 1997). Seeds were sown on filter paper saturated with 1-aminocyclopropane-1-carboxylic acid (ACC) at 0, 0.01,

0.05, 0.1, 0.5, 1, 5, 10, 50, or 100 μM. Hypocotyl length was measured at 12 d after germination. Bars represent the percent of hypocotyl length as compared to the wild type

(0 μM ACC) for each concentration, error bars represent the standard error, and letters above error bars represent significance between treatments based on least squares means test using α = 0.05 for significance. (A) Petunia × hybrida ‘Mitchell Diploid’, (B)

Petunia × hybrida ‘44568’ ert1-1.

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Figure B.1. continued

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Appendix C: Illustration of how leaf angles were taken in Chapter 3.

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Figure C. 1. Illustration of how leaf angles were measured in Chapter 3. Leaf angles were obtained for the bottom six leaves per plant by measuring the angle between the stem and the top of the petiole. These six values were used to calculate the average epinastic curvature per plant. Measurements were taken until leaf angles were within 10° of the initial angle measurements and plants were considered to be recovered.

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