1 MODE OF ACTION OF AN ABSCISSION AGENT AND A MODEL TO PR E DICT LOOSENING OF SWEET ORANGES AS AN AID TO MECHANICAL HARVESTING By SUNEHALI SHARMA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA I N PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2013
2 2013 Sunehali Sharma
3 To the Divinity that exists within all of us
4 ACKNOWLEDGMENTS This the sis is a product of a team of people. I have been fortunate to have Dr. Bob Ebel as my primary advisor. I would like to extend sincere thanks to him for his expert guidance and constant encouragement throughout my doctoral program. Not only he has shaped m y academic career, his personal philosophy towards life has greatly influenced my personal growth. He has been a wonderful mentor and a joyous person to work with. I would like to thank my dissertation committee members, Dr. Mark Ritenour, Dr. Ed Etxeberri a, and Dr. Kelly Morgan for their valuable guidance and constructive criticism. Their comments had a huge, positive impact on the final version of this thesis. Along the way, I was lucky to have Dr. Naveen Dixit in the team. I would like to express my sin cere gratitude to him for helping me in building a strong foundation of scientific skills needed for my research work. He has constantly supported my efforts to Newman, Biological Scientist, who helped me a great deal in setting up my experiments. I have enjoyed all our time working together and this had a positive effect on my research. m ade this whole journey enjoyable for me. The most important thanks go to my mother, Rekha Sharma. Despite all the personal hurdles, her continued patience has never have been able to complete this job without the blessings of my father, Varinder Kumar Sharma, who never left my side till the end
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 LITERATURE REVIEW ................................ ................................ .......................... 12 Challenges Leadi ng to Emergence of Mechanical Harvesting ................................ 12 Research and Development of Mechanical Harvesting of Sweet Orange in Florida ................................ ................................ ................................ .................. 13 History ................................ ................................ ................................ .............. 13 Types of Mechanical Harvesters ................................ ................................ ...... 14 Obstacles in Adopting Mechanical Harvesting ................................ ........................ 16 Benefits of Using CMNP as an Abscission Agent ................................ ................... 19 Factors Affecting Efficiency of CMNP ................................ ................................ ..... 21 Application Factors ................................ ................................ ........................... 21 Climatic Factors ................................ ................................ ................................ 23 Plant Based Factors ................................ ................................ ......................... 24 Physiological Effects of CMNP ................................ ................................ ............... 25 Alternative Hypothesis for Signaling Mechanism of CMNP in Sweet Orange ......... 30 Nitro Substituted Pyrazoles Produce NO in Living Tissue ................................ 30 Sources, Role and Fate of NO in Biological Organisms ................................ .......... 31 Sources of NO in Plants ................................ ................................ ................... 31 R ole of NO in Plants ................................ ................................ ......................... 32 Fate/Removal of NO ................................ ................................ ......................... 33 Objective of This Thesis ................................ ................................ .......................... 34 2 ROLE OF NITRIC OXIDE IN ABSCISSION OF SWEET ORANGE ....................... 37 Material and Methods ................................ ................................ ............................. 40 Results and Discussion ................................ ................................ ........................... 42 Fruit Detachment Force of SNP and CMNP Treated Fruits .............................. 42 NO, NO 2 and NO 3 Concentrations after CMNP Treatment ............................. 43 3 INVOLVEMENT OF ETHYLENE IN ABSCISSION OF SWEET ORANGE ............. 49 Materials and Methods ................................ ................................ ............................ 51 Data Collected ................................ ................................ ................................ ........ 52
6 Results and Discussion ................................ ................................ ........................... 54 FDF of Fruit Treated with AVG and STS in Preliminary and Second Experiment ................................ ................................ ................................ .... 54 Effect of CMNP and AVG on FDF ................................ .............................. 54 Ethylene production and its effect on FDF ................................ ................. 55 Effect of CMNP and STS on FDF ................................ .............................. 57 Ethylene production and its effect on FDF ................................ ................. 57 4 A PREDICTIVE MODEL FOR PROMOTING ABSCISSION OF SWEET ORANGES AT VARYING TEMPERATURES BY AN ABSCISS ION AGENT ......... 68 Material and Methods ................................ ................................ ............................. 69 Results and Discussion ................................ ................................ ........................... 72 Model Deve lopment ................................ ................................ ......................... 72 Model Validation ................................ ................................ ............................... 74 5 FUTURE RESEARCH AND SUGGESTIONS ................................ ......................... 80 Mode of Action ................................ ................................ ................................ ........ 80 Empirical Model ................................ ................................ ................................ ...... 81 LIST OF REFERENCES ................................ ................................ ............................... 82 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 100
7 LIST OF TABLES Table page 1 1 Some effects of exogenous applications of NO via SNP on plants. .................. 35 3 1 Treatment of fruit with AVG, STS and CMNP in the first experiment ................. 62 3 2 Treatment of fruit with AVG, STS and CMNP in second experiment. ................ 63
8 LIST OF FIGURES Figure page 1 1 Physiological effects of CMNP in sweet orange. ................................ ............... 36 2 1 and control fruit treated with water. FDF data was expressed as means SE from 10 fruits per treatment. ................................ ................................ ............... 46 2 2 the abscission agent CMNP and water (control). Means within each sampling time followed by different letters indicate significant difference at P < 0.05. ....... 46 2 3 treatment with the abscission agent CMNP. Means within each sampling time followed by different letters indicate significant difference at P < 0.05. ............... 47 2 4 NO 2 treatment with abscission agent CMNP. Means within each sampling time followed by different letters indicate significant difference at P < 0.05. ............... 47 2 5 NO 3 treatment with abscission agent CMNP. Means within each sampling time followed by different letters indicate signifi cant difference at P < 0.05. ............... 48 3 1 Fruit detachment force of fruit treated with CMNP at 2 mM and inhibitors at different concentrations in preliminary experiment. Difference in means was shown ................................ .................... 64 3 2 Fruit detachment force of fruit treated with CMNP at 2 mM and inhibitors at different concentrations in second experiment. Difference in means was ................................ .................... 65 3 3 Ethylene abscission agent CMNP and the ethylene synthesis inhibitor AVG. E thylene data was expressed as means SE from 12 fruits per treatment. Bars not visible lie within the height of the data symbol. ................................ ................... 65 3 4 Fruit detachment force (FDF) correlated with the total ethylene flux over the significant to the P = 0.02 level. ................................ ................................ .......... 66 3 5 r treatment with abscission agent CMNP and STS. Ethylene data was expressed as means SE from 12 fruits per treatment. ................................ ................................ .......... 66
9 3 6 Fruit detachment force (FDF) correlated with the total ethylene fl ux over the significant to the P = 0.35 level. ................................ ................................ .......... 67 4 1 Goodness of fit of a logistic function to fruit detachment force as affected by air temperature. The function was fitted to data of Yuan and Burns (2004). ...... 77 4 2 ully mature trees in a commercial grove and spraye d at two concentrations of CMNP ................................ ................................ ......... 78 4 3 dates during the coldest parts of the winter months when this cultivar is normally harvested in Florida.. ................................ ................................ ............ 79
10 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requiremen ts for the Degree of Doctor of Philosophy MODE OF ACTION OF AN ABSCISSION AGENT AND A MODEL TO PR E DICT LOOSENING OF SWEET ORANGES AS AN AID TO MECHANICAL HARVESTING By Sunehali Sharma December 2013 Chair: Robert C. Ebel Major: Horticultural Sciences Rigorous effort ha s been made in the past few decades on research and development of mechanical harvesters and finding a suitable abscission agent for use in citrus as an aid to mechanical harvesting. Various studies had been conducted and CMNP (2 chloro 3 methyl 4 nitro 1H pyrazole) was recognized as a potential abscission agent to be used in citrus. The mode of action of CMNP in sweet orange is largely un known the knowledge of which would be valuable in developing recommendations for its use in commerci al industry Based on the structure of CMNP and the way sodium nitroprusside (SNP) also caused ab s ci s sion it was hypothesized that nitric oxide could be the signal involved in abscission by CMNP. Concentrations of nitric oxide produced inside fruit after CMNP application were in the nanomolar range, yet millimolar concentrations were required to promote abscission when SNP was used The concentrations of NO found are typical in plant species to promote healing from physiological stress. Although some res earch had been previously conducted on the role of ethylene in promoting abscission, a more thorough approach was conducted to clarify its role. The ethylene synthesis inhibitor aminoethoxy vinyl glycine (AVG) and the ethylene receptor binding inhibitor s ilver thiosulfate (STS) were evaluated on CMNP
11 treated fruit AVG promoting abscission. Results from STS were complex to interpret because it promoted e thylene evolution independent of its toxic concentration and its p oisoning effects on metabolism. Nevertheless, the promotion of loosening by CMNP was less in fruit pretreated with STS. The results indicate that the promotion of abscission by CMNP involves ethylene. From these data, a mech anistic model is hypothesized that involves the creation of 1 Aminocyclopropane 1 carboxylic acid under the anoxic conditions promoted by CMNP which moves to the abscission zone and promotes abscission. Since the mechanism of CMNP is not fully resolved, a mathematical model that predicts loosening based on the mechanism cannot b e developed. Therefore, an empi rical model was developed from data published in the literature and verified using data from fruit. Further testing of the model is needed before it could be used commercially in the event that CMNP is registered by the EPA.
12 CHAPTER 1 LITERATURE REVIEW Challenges Leading to Emergence of Mechanical Harvesting The total worldwide production of orange s in 2009 10 was of 49,360 metric ton s (FASS, 2011 ) During this period, Brazil ranked first in orange production amongst citrus producing countries followed by the United States. In the United States, Florida is th e major producer of citrus contributing 71% to the total production followed by California accounting for 26%, with Arizona and Texas producing the majority of the rest Florida produced 6012 metric tons of oranges in 2009 10 95% of which is processed int o juice (FASS, 2011 ). Florida and Sao Paulo state are the two most important juice processing regions in the USA and Brazil respectively and accounted for 53.7% of total global orange juice production (FASS, 20 11 ). Citrus acreage and processing capacity o f Brazil expanded during 1970s and 1980s, while, Florida citrus industry suffered a major setback s due to severe fr eezes, allowing Brazil to emerge as the producer in 1983 (Tetra Pak, 1998). Citrus production and processing indust ries in Florida have grown substantially over the y ears to meet consumer demand However, i ncreasing harvesting costs, shortage of labor, and disease and pest infestations are among the major challenges that have threatened citrus growers economically. The cost of h and harvest and hauling oran ges to processing plants exceed production costs in Florida. Production cost s have increase d to promote recovery from hurricanes citrus canker and greening diseases. More than 70% of harvest labor workers are foreigne rs and working without legal documentation (NAWS, 2005). Recent immigration reforms have intensified the concerns over labor availability during the citrus harvest season (Sparks, 2008). Long
13 term solutions for HLB and citrus canker diseases have not been found. Until solutions are developed growers must implement new production strategies to maintain economic viability. Mechanical harvesting has evolved as one of the solutions to reduce overall production costs to sustain the citrus industry economically. However concerns about the impact of mechanic al harvesters on tree health has resulted in a reduction in mechanically harvested acreage in recent years. Research and Developme nt of Mechanical H arvesting of Sweet Orange in Florida History Research and dev elopment in mechanical har vesting dates back to the 1950s. H owever, interest in this area has not been consis tent over the decades, escalating from 1960 1970, diminishing in mid 1980 and increasing again after 1990. In 1957 s carce labor supply and expand ing acreage in Florida led to establish ment of a citrus harvesting research project at U niversity of Florida CREC, Lake Alfred. Finding a viable mechanical picker was the main focus of many projects during the 1950s, 60s, and 70s. In the early 1960s, con tact devices were investigated which showed little potential for acceptance. In the late 1960s, research was directed towards development of mass removal harvest systems which appeared to have greater potential to harvest oranges for processing industries. These included limb, trunk, foliage, and air shakers. technology and hence, federal funding for mechanical harvesting research ceased ( Roka et al., 2009) Funding stopped altog ether by the early 1980s due to a series of devastating freezes. Successive freezes led to expansion of citrus acreage in southwest Florida whi ch is less prone to freezes but interest in mechanical harvesting did not develop again until the late 1990s
14 T ypes of Mechanical Harvesters Contact devices were developed in the early 1960s and different type s of shakers in 1970s. Research and development for new machinery continued until a suitable mechanical picker was designed that would provide adequate fruit removal. Contact devices Contact devices were designed to duplicate the fruit separation techniques of manual harvesters. Such devices included rotating rubber rollers and cones (Coppock, 1971), flexible hooks (Pictiaw, 1973), vacuum cups (Schertz and Bro wn, 1968), and rubber spindles (Coppock, 1961). Fruit removal efficiency of 65 to 75% could be achieved from trees having heights less than 4 m. Shakers Several types of shakers were investigated in the past few decad es. L imb, foliage, and trunk shakers removed fruit by directly contacti ng various parts of the tree, while air shaker s caused fruit removal without making any direct contact. Limb shakers apply force to scaffold branches to accomplish fruit removal. A five year study by Hedden and Coppock ( 1965) revealed that limb shakers did not affect yields of early and mid season citrus, while yields of late se 40% per season. Foliage shakers remove fruit by applying force to the outer parts of the canopy Removal of only matu Mature fruit removal averaged 85% with a foliage shaker and 88% with a limb shaker over two seasons of harvesting trials (Hedden and Coppock, 1971). Trunk shakers ha d been utilized in Florida for about three decades as fruit removal d evices. Trunk shakers had been initially used in California for nut and prune harvesting and were modified for use on sweet oranges in Florida. T runk shakers were
15 rejected for sweet oranges because of bark damage, low fruit removal and lack of adequate tree trunk area for shaker clamp attachment. Air shakers remove fruit by pulsating a high velocity air stream into the tree. In the w ere greater in early and mid season varieties (Whitney and Patterson, 1972), while 15 40% reduction was observed in Valencia orange when the fruit diameter reached 3 cm. The main advantage of air shaker s over limb, foliage, and trunk type of shakers is tha t no physical contact was made to tree. Operational reliability of air shaker was found higher than that of limb and foliage shakers. However, air shakers had a high power requ irement and initial investment s and were therefore rejected for commercial use. Current commercial harvesting s ystems There are two kinds of canopy (TCS) are pulled behind a tractor while dropping fruit to the ground (Whitney, 1977). Since there is n o catch frame, trees do not have to be skirted or pruned and fruit must be picked up by hand from the ground (Bora et al., 2006; Hedden et al., 1983; Whitney, 1999). Second, self propelled canopy shakers or continuous canopy shake and catc h (CCSC) harvest ing system s work in pairs on both side of a tree with the decks positioned under the tree to catch the fruit while moving in unison down the row. Trees have to be skirted and pruned to the lower scaffold limb to avoid unnecessary damage to the tree and to maximize efficiency of catch frames. Removal rates varied from 50 98% in research studies (Whitney, 1975, 2000, 2003; Whitney et al., 2001).
16 Despite aggressive effort to develop and adopt mechanical harvesting technologies and their advantage s over conven tional hand harvesting, the transition towards mechanical harvesting has been relatively slow. Obstacles in Adopting Mechanical Harvesting Widespread adoption of mechanical ha rvesting has been impeded by grower s concerns over physical injury caused by mac hine s during removal of young fruitlets of Valencia during the Tree i njury M echanical harvester s currently used commercially can cause physical injur y to the trees, including broken branches, shedding of leaves flowers, and young fruits, and scuffing of bark (Halderson, 1966; Buker et al., 2004; Whitney, 2003) Research on the effects of mechanical harvesting on tree physiology has shown no impact on sufficiently healthy trees to withstand the impacts of mechanical harvesting without reduction in yields (Hedden and Coppock, 1968; Li and Syvertsen, 2005; Li et al., 2006; Yuan et al., 2005; Whitney et al., 1986). However, the decline in adop tion of mechanical harvesting has been due to citrus greening a bacterial disease that h as been perceived to make trees too weak to handle the additional stress imposed by mechanical harvesters. Late Another impediment towards adopting mechanical harvesting is the problem brought about harvesting simultaneously February/March and harvesting begins in March of the following year and continues thro ugh June and sometimes into July. By mid size and weight to be susceptible for removal by mechanical harvester s Harvesting
17 should be terminated when Valencia fruit are 1 inch in diameter when an abscission agent i s not used (Coppock, 1972). Labor availability Concerns about s carcity of labor are the primary motivation by growers to develop and implement mechanical harvesting in commercial groves However, due to their fear of tree injury and acceptable f ruit rem oval, growers are more inclined towards hand harvesting when labor is available in abundance. It is considered by the research community and the commercial citrus industry that an effective abscission agent that selectively loosen s with minimal tree injur y would promote expansion of commercial acreage mechanical ly harvest ed, especially during labor shortages Abscission Chemicals as Mechanical Harvesting Aids Researchers have investigated various compounds in different field studi es and have evaluated each compound for meeting an important set of criteria needed to be considered as a potential candidate for registration. Abscission chemicals have been used as an aid to mechanical harvesting in loosening many fruit crops, including citrus (Bukovac et al., 1969; Bukovac, 1979; Lavee and Haskal, 1975; Perez et al 1981; Wilson et al 1981). From the mid 1960s to mid 1980s researchers with the Florida Department of C itrus (FDOC) and United States D epartment of Agriculture (USDA) eval uated many abscission compounds that could be used for loosening citrus (Kender, 1998; Wilson et al., 1977, 1981). Among the se were Acti aid (Cycloheximide), ethephon (2 chloroethylphosphoric acid), PIK OFF (glyoxal dioxime), and Release (2 chloro 3 methyl 4 nitro 1H pyrazole, i.e., CMNP). None of the abscission agents were commercialized at that time due to ample labor and severe freezes during the 1980s.
18 In 1995, with the financial support of the FDOC, scientists at the CREC in Lake Alfred tested sever al new compounds such as metsulfuron methyl, prosulfuron, methyl jasmonates, and older compounds including, CMN pyrazole (Release) and ethephon (Hartmond et al., 2000 ). Effects of metsulfuron methyl and prosulfuron were studied on 4 mg/L metsulfuron methyl. The symptoms became more severe as fruit loosening increased (Kender et al., 1999). Pros ulfuron was Moreover, it induced excessive drop of immature fruit and induced p hytotoxicity (Kender et al., 1998; Whitney, 1998) individual fruit or as a spray to entire trees. Methyl jasmonate at 10 mM and lower did not cause p hytotoxicity and lowered fruit detachment force ( FDF ) C oncentrations of 20 and 100 mM in duced unacceptable levels of leaf abscission (Hartmond et al., 2000). Another study by Kender et al respond to 10 mM methyl jasmonate and required 20 mM methyl jasmonate to cause loosening to below FDF of 50 N, but it resulted in excessive leaf loss. Ethephon concentrations of 200 500 mg/L resulted in good fruit abscission but caused excessive defoliation (Kender et al., 2000b).
19 Application of CMNP at 200 300 mg/L red uced FDF up to by 90% 4 days a f ter (Davies et al., 1975; Wheaton et al., 1977; Burns, 2002) but caused defoliation above 1000 mg/L (Burns, 2002). Criteria for e valuation for an abscission agent According to Kender and Hartmond ( 2000) a suitable abscission compound must: Be selective in its action Have minimum effect on tree health and yield Not have phytotoxic effects Be approved by EPA for registration Resea rch has shown that CMNP is the only known potential candidate that me e t s the above criteria except the last one and therefore has been chosen by the Florida citrus industry to attempt registration by the EPA CMNP enhances the abscission of only mature Val encia fruit without affecting yield of the subsequent has been shown to cause no phytotoxic ity on leaves or young fruits (Li et al., 2008). Benefits of U sing CMNP as an Abscission A gent Use of a sel ective abscission agent with mechanical harvester s can boost operational efficiency of a mechanical harvesting system by extending the harvest window for mechanical harvesters, operating faster in the grove, improving recovery percentages of fruit, and red ucing harvest cost. Increased h arvest speed Without CMNP use canopy shakers travel at 0.75 1.25 miles/ hr spending 10 sec/tree. W ith the use of CMNP, harvesters speed can be increased to 2 miles/hr, spending half the amount of time in a tree yet removin g the same amount of fruit (Burns et al., 2005).
20 Reduce tree injury It is thought that the l esser time required to harvest each tree after CMNP use would translate into reduce tree injury Operating equipment at lower intensities would also lessen the me chanical stress and reduce equipment repair cost as well With the use of CMNP and less aggressive shaking, the load of twig and leaf debris transported to the processing plant would also be reduced (Spann, 2007). L ate season Valencia p roblem Citrus gen erally blooms in Feb M ar and Valencia fruit takes 12 14 months to mature. Thus there are two crops present on the same tree at harvest, a mature crop and the immature crop. CMNP selectively loosen the mature crop and does not cause abscission of the immat ure crop and leaves (Holm and Wilson, 1977; Kender, 1998; Wilson, 1973) If immature fruit have diameter s greater than 1 inch, they are reported to be removed during mechanical harvesting and cause 25% yield loss in next season crop (Whitney, 1975). With u se of CMNP on mature fruit and 1 inch diameter young fruit, harvesters shaking settings could be scaled back and thus crop will be less adversely affected (Burns et al 2006). Fur ther, use of an abscission agent extends the harvest window by 2 3 weeks and increases the number of boxes harvested (Roka and Burns, 2007) This would decrease harvesting costs by spreading the cost over greater no. of boxes harvested in one year. Solve l abor issues Higher labor costs, cumbersome immigration reforms, and uncertainty of enough labor availability during the late season harvesting period have always been a concern to citrus grower s (Ebel et al., 2011) Use of CMNP along with mechanical harve sters has been shown to decrease the nu mber of labor worke rs required to harvest the crop and increased returns per tree.
21 Factors A ffecting Efficiency of CMNP Responsiveness of citrus fruit to CMNP is dependent on application, climatic and plant based fact ors. Application F actors Research has already shown that CMNP efficacy is largely a function of concentration, coverage, and uptake of CMNP (Alferez et al ., 2005; BenSalem et al ., 2001; Burns et al ., 2006; Ebel and Burns 2008; Farooq et al ., 2003; Kender and Hartmond 1999; Koo et al ., 1999, 2000; Salyani et al ., 2002). CMNP concentration A linear response of concentration on FDF between 0 to C. sinensis X Poncirus trifoliata (L ) Raf.] rootstock was found by Burns et al (2005). However, no research has been done to report linear response between FDF and CMNP concentration in Valencia. The linear response allows selection of a concentration that would give a desirable reduction i n FDF for maximum fruit removal, which is about 50% of the starting FDF for ( Burns et al ., 2005) Concentrations above 250 ppm have reported to cause yield reduction of 77). Application of CMNP at 50 and 100 mg/ml a.i. before and after the period following blossom) in Valencia resulted in a 10% to 12% increase in fruit removal with a trunk shake and catch harvesting system when compar ed to control trees. Only 100 mg/ml a.i. CMNP significantly increased fruit removal in Valencia orange when et al ., 2000). T he harvesting season for Valencia fruit usually begins in March and extends th rough May. The response of mature Valencia fruit to abscission chemicals seems to be inconsistent
22 (Cooper and Wilson, 1971; Hartmond et al ., 2000 ; Holm and Wilson, 1977; Wheaton et al ., 1977) starting from late April to early May, and lasting for 2 to 6 w eeks (Hartmond et al ., 2000 ; Holm and Wilson, 1977; Wheaton et al et al ., 2000 ). CMNP volume/spray volume Linear relationships between CMNP volume and the per cent coverage and FDF wer e found for sweet orange (Ebel et al 2009 ) Any adjustment in volume applied per hectare will result in a linear response in coverage and fruit loosening (Ebel et al 2009). Salyani (1999) defined deposition efficiency as deposition (g/cm 2 ) per unit d ischarge duri Applying et al (1999) found that l ower spray volumes resulted in higher deposition than higher volumes. However, more uniform deposition, lower fruit detachm ent force, and higher percent fruit removal were observed with higher volume spray (Koo et al., 1999) With CMNP, full coverage of the tree is required since direct contact with the peel is necessary to promote loosening (Ebel et al ., 2009). It was shown that CMNP application with in 1 cm to abscission zone promote loosening indicating signal might travel from peel to abscission zone (Burns et al., 2006). In general, 300 gal/acre are the suggested volumes of solution to use when applying CMNP. Uptake and distribution of CMNP I t is believed that the effectiveness of CMNP is directly related to the amount of active ingredient actually contacting and penetrating the orange peel (Winkler et al ., 1974). Kossuth et al (1978) recovered 78 to 94% of 14 C labeled CMNP in the flavedo, 5 19% in the albedo tissue, and less than 1.5% in the pulp. Wheaton et al (1977) observed better uptake of 14 C CMNP in immature fruit than
23 in mature fruit. Several physiological and structural factors may account for this observation. fruit have a thinner wax layer than mature fruit and more stomata per unit area (Albrigo, 1977). Nevertheless, although more CMNP is taken up by immature fruit, it does not promote abscission of immature fruit indicating a differentia l sensitivity compared to mature fruit. Climatic Factors Many plant features and climatic factors have been identified that may affect efficacy of CMNP. Climatic factors include temperature, relative humidity, and precipitation during the first eight hou rs after application (Biggs and Kossuth, 1980; Kossuth et al ., 1978; Yuan and Burns, 2004). CMNP loosens mature fruit within 3 to 5 days depending upon temperature o C is considered a critical minim um below which activity of CMNP slows and rate of loosening declines (Ebel and Burns, 2008). FDF was not affected by application of CMNP at 200 mg/L when air o C (Yuan and Burns, 2004). Peel uptake of 14 C CMNP has been shown to var y w ith t emperature and relative humidity (RH). Peel uptake is maximum at 58 to 66% RH and 25 o to 35 o C (Kossuth et al ., 1978) while minimum uptake was observed at low temperature and high RH did not improve uptake rate at low temperature (Murphy et al., 1976 ) Kossuth and Biggs (1978) reported that s imulated or true rain removed 70% or more of the 14 C CMNP during the 0 8 hour period after peel application in the field, but a 24 hour rainless period resulted in recovery of up to 66% of the compound from the fr uit (Murphy et al ., 1976; Kossuth et al ., 1978). Because of the adverse effects of rain, CMNP use may be limited during rainy periods, especially after the rainy season starts.
24 Plant Based Factors Aspects of plants that may affect CMNP efficacy include sta ge of crop maturity, type of rootstock and cultivar, and tree health (Biggs and Kossuth, 1980; Hartmond et al., 2000; Yuan et al., 2001 ). Stage of crop maturity CMNP applied at 300 ppm promoted higher reduction in during late January compa red with December and early January as a result of over maturation (Ebel et al., 2010). F DF f luctuat es throughout the Valencia harvest season (Holm and Wilson, 1977), indicating that fruit maturity may play a role in response to abscission chemical s Howe ver, several other physiologica l factors may contribute toward the which generally occurs in May in Florida Kender et al (2001) conducted a seven month study from early November 1998 to mid June 1999 on Valencia orange and showed that IAA concentration in peel and the abscission zone was higher and ABA concentration was DF of CMNP treated fruits was positively correlated with the endogenous IAA/ABA ratio and negatively c orrelated to ABA in the abscission zone during, thereby, indicating that IAA and ABA are the essential aspects determining response in Valencia to any abscission compound during a particular growing stage. Type of rootstock and cultivar o loosen faster (Sharma et al. 2010) possible since their harvest periods are different in Florida. No research has been conducted to determine if rootstock impacts CMNP efficacy. However, many field trials have been conducted since the mid 1970s on several rootstocks and it appears that CMNP performs consistently on all rootstocks tested to date.
25 Tree health S tress likely has some effect on CMNP efficacy h owever, short term dro ught stress appeared to have little effect on the rate of loosening (Ebel et al., 2008) How diseases may impact its efficacy is not known. HLB, which is known to promote abscission and fruit drop of infected trees, will likely alter CMNP efficacy altho ugh no research has been performed to date to address this particular question. Hence, the efficacy of CMNP can be affected by external and internal factor s. After uptake by the fruit, CMNP alters metabolism in a manner that promotes a bscission. Understan ding the factors that affect the mode of action of CMNP is required before an efficient mathematical model to predict loosening can be developed. Such a model would have utility by harvest managers in the commercial industry for scheduling CMNP applicatio ns and harvest Physiological Effects of CMNP Some aspects of the mode of action of CMNP ha ve been determined but the mechanism is still largely unknown. CMNP has a pyrazole backbone with methyl, nitro, and chlorine groups substituted at the 3, 4, and 5 p ositions, respectively, on the ring ( 1 1). The nitro and chlorine groups have lower electro negativity that creates polarity with the electron cloud favoring that side. (1 1)
26 Known metabolic activity of CMNP in sweet o range Microarray analysis in Arabidopsis revealed that CMNP regulates genes associated with stress, anoxia, and senescence ( Alferez et al 2007). CMNP applied to flavedo tissue within 1 cm of the abscission zone promoted abscission (Burns et al 2006), indicating that CMNP eith er moves to the abscission zone or evokes a signaling process in flavedo tissue from which a signal moves to the abscission zone and activates the abscission process. The metabolic pathways known to be affected by CMNP include lipid signaling, accumulatio n of acetaldehyde, and uncoupling of ATPase activity (Figure 1 1 ) As CMNP moves into the cell, it must first cross the plasma membrane which contains enzymes involved in lipid signaling, although it will also move across other membranes as it traverses t he cell. From the plasma membrane it moves into the cytosol affecting ADH activity and then into the mitochondria where it uncouples ATP activity. The latter Alferez et al 2007). Increase in ethylene c oncentration has also been reported till day 3 after CMNP application (Yuan and Burns, 2004). Lipid signaling To enter the cell, CMNP must first cross the plasmalemma, which contains several enzymes involved in lipid metabolism. Lipid signaling would be further enhanced as CMNP moves through other membranes throughout the cell. Lipoxygenase (LOX) and phospholipase A 2 (PLA 2 ) activities and lipid hydroperoxide (LPO) content were shown to increase in citrus flavedo tissue after CMNP treatment ( Alferez et al 2005). The generation of membrane degrading products is an indication of involvement of the lipid signaling pathway (Wang et al 2002; Meijer and Munnik, 2003). Phospholipase D (PLD) gene expression was shown to be upregulated in citrus
27 peel tissue 24 expression was diurnally regulated in fruit peel with peak expression around midday (Malladi and Burns, 2008). Further, CMNP was found to induce expression of genes similar to allene oxide syn thase and 12 oxophytodienoate reductase in the mature fruit abscission zone (Burns, 2002) indicating potential involvement of Jasmonic acid in the abscission process. In general, products of lipid signaling accumulate under abiotic and biotic stresses and promote senescence (He et al 2002). Although jasmonic acid is a known inducer of senescence and abscission (Hamberg and Gardner, 1992; Vick and Zimmerman, 1987), exogenous applications of methyl jasmonate did not induce abscission of mature sweet orange (Kender et al 2001). Thus, the role of lipid signaling in promoting abscission of mature citrus is not clear. It should be noted that application of CMNP at higher concentrations does promote senescence of flavedo tissues. Acetaldehyde accumulation From the plasmalemma, CMNP moves into the cytosol where it inhibits ADH activity ( Alferez et al., 2005). Inhibition of ADH by pyrazole and other substituted pyrazoles have been known for several decades (Dahlbom et al., 1974). The mechanism by which CMNP inhibits ADH activity is unclear, but likely involves its binding to NAD + (Li and Theorell, 1969). By inhibiting ADH, acetaldehyde cannot be converted to alcohol and thus accumulates. Furthermore, CMNP promotes gene expression of pyruvate decarboxylase ( PCD1), promoting accumulation of acetaldehyde and causing electrolyte leakage within 2 hr after CMNP application ( Alferez et al 2006). The higher respiration and depleted carbohydrate pool promotes expression of ADH1 ( Alferez et al 2007 ; Leverentz et al., 2002 ), which is the
28 form of ADH found in the cytosol. Initial depletion of soluble carbohydrates is compensated by expression and activity of the pathways associated with these enzymes and results in higher soluble carbohydrates 72 hr after CMNP treat ment ( Alferez et. al., 2006). Uncoupler of ATPase activity Nitro substituted compounds have been shown to uncouple ATPase activity that reduces ATP content (Hanstein, 1976), which is likely by the direct ferrying of protons across membranes (Chopineaux C ourtois et al 1999). The polarity of the nitro group would allow protonation of an oxygen moiety. CMNP has been shown to decrease ATP content in flavedo tissues of sweet orange, which promotes acceleration of respiration and oxygen consumption ( Alferez et al 2005). Ethylene production and up regulation of cell wall enzymes Typically, abscission is accompanied by a simultaneous increase in ethylene concentration and synthesis of hydrolytic enzymes, cellulase and polygalacturonase in the abscission z one that culminates in degradation of the cell wall, and finally separation of the vascular tissues (Goren, 1993; Kazokas and Burns, 1998). Ethephon promote s fruit loosening by producing ethylene upon its own degradation and CMNP also stimulates ethylene e ggs, 1977). Ethylene evolution by ethephon and CMNP treated fruit is temperature dependent. FDF decreased drastically and ethylene evolution increased in ethephon treated and CMN P treated fruits as temperature increased from 10 o C to 26.7 o C and 15.6 o C to 26.7 o C for ethephon and CMNP respectively (Yuan and Burns, 2004) E thylene production started increasing after 40 hours of CMNP application and it peaked at 81 hours after applicat ion (Kossuth and Biggs, 1977).
29 Ethylene hastens appearance and action of cellulase and other cell wall hydrolases (Brummell et al., 1994; Sexton and Roberts., 1982). CMNP also induces and Biggs, 1977). Besides similarity in behavior and course of action of CMNP and ethylene, t here are f ew studies on CMNP metabolism that suggest that abscission caused by this abscission agent is ethylene independent. In o ne study by Bur ns et al (2006) CMNP caused a transient increase in soluble sugar content, however, ethephon had little effect on soluble sugar content and starch reserves. Another study confirmed that ethylene applied in the form of ethephon was shown to induce absciss ion of mature sweet orange (Yuan et al 2001), but it also caused leaf abscission, a phenomenon not observed with CMNP (Holm and Wilson, 1977; Kender, 1998). Furthermore, use of the ethylene inhibitor, 1 MCP, in combination with CMNP did not stop reductio n in FDF in Pozo et al 2004). From these findings, Alferez et al (2006) concluded that CMNP and ethylene act independently to accelerate mature fruit abscission. In summary, previous studies demonstrate that CMNP produced anoxi a like conditions, but the mechanism by which this phenomenon promoted abscission has not been conclusive. Jasmonate and ethylene, two compounds known to promote abscission in other species, appear not to be the critical signals involved in abscission in sweet orange. Thus, CMNP can be applied solely to flavedo tissue within 1 cm of the abscission zone and cause abscission, but the signal produced through the activity of CMNP that moves to the abscission zone is unknown.
30 Alternative Hypothesis for Signa ling M e chanism of CMNP in Sweet O range An alternative hypothesis by which CMNP causes abscission is based on previou s studies on nitro pyrazoles This hypothesis proposes that CMNP produces nitric oxide (NO) and that NO either directly or through promotion of another signal causes abscission in sweet orange (1 2 ) (1 2 ) Nitro S ubstituted P yrazo les P roduce NO in Living T issue N itro substituted pyrazoles have been evaluated for years as potential treatments for diseases involving poor oxygen transport in eyes of mammals (Xuan and Chiou, 2003). To promote blood flow and thus improve oxygen movement into eyes, compounds have been sought that produce NO, which relaxes smooth muscles such as those lining arteries. Several studies have shown that nitro substituted pyrazoles produce NO (Xuan and Chiou, 2003). Many nitro substituted pyrazoles have been studied and all produce NO, alth ough to varying degrees depending on where the substitution occurs on the pyrazole ring (Xuan and Chiou, 2003). It should be noted that these pyrazoles have the nitro substitution on one or both of nitrogens in the pyrazole ring whereas in CMNP the nitro is substituted on one of the carbons in the ring. Nitro group substituted at nitrogen or carbon have been shown to produce NO in different amounts.
31 Sources, Role and Fate of NO in Biological Organisms Sources of NO in P lants L arginine dependent pathway Nitric oxide synthase (NOS) has been shown in many studies to be involved in production of NO (1 3 ) NO and its substrate L arginine have been reported in several plant tissues and purified organelles, including mitochondria, nucleus, and peroxisome (Barro so et al 1999; Corpas et al 2006). Oxygen and Ca 2+ /calmodulin are obligatory for the reaction to occur. L arginine + NAD(P)H + O 2 L citrulline + NAD(P) + + H 2 O + NO (1 3 ) L arginine independent pathways There are also L arginine in dependent pathways that produce NO. These pathways include reduction of nitrite via: Cytoplasmic nitrate reductase (cNR) Nitrate reductase normally catalyses reduction of nitrate to nitrite. However, under certain circumstances such as anaerobic conditio ns which results in nitrite accumulation (Dean and Harper, 1988), it has been shown to produce nitric oxide from nitrite as well (Rockel et al 2002). PM NR/Ni:NOR Plasma membrane bound nitrate reductase reduces nitrate to nitrite in the apoplasm that ma y further be reduced to NO by NO oxidoreductase. Mitochondria Under anoxic conditions, pure mitochondria was shown to produce NO when supplied with nitrite and NADH (Gupta et al 2005). Xanthine oxidase/dehydrogenase (XDH) XDH has been suggested as a so urce for NO having xanthine and nitrite as substrate (Millar et al 1998). Non enzymatic NO production occurs at low pH (below 4.5) in apoplast of plant cells (Bethke et al., 2004)
32 Role of NO in Plants NO has diverse roles in higher plants depending on i ts concentration (Table 1 1 ). Lower concentrations of either endogenously produced or exogenously applied NO in the 10 6 M range exert growth promoting effects which can be reversed by higher NO conc entrations (Leshem, 1996; Belig ni and Lamattina, 1999). N O has an essential role in inhibiting catalase, ascorbate peroxidase, Cytochrome c oxidase, and aconitase activities (Clark et al 2000), regulation of ion channels in guard cells (Garcia Mata et al 2003), cell death (Pedroso et al 2000; Saviani et al 2002), wound signaling (Orozco Cardenas and Ryan, 2002), mitochondrial and chloroplastic functionality (Yamasaki et al., 2001; Zottini et al., 2002), iron homeostasis (Murgia et al 2002), and senescence (Leshem, 1996). NO may have anti senescence pro perties by interrupting chain reactions leading to lipid peroxidation, thus, preserving photosynthetic pigments and having a positive effect on chlorophyll biosynthesis (Jasid et al 2009). NO also prevents formation of most deleterious ROS, the hydroxyl 1995). Fumigation of NO @ 0.25 mol delayed senescence and prolonged post harvest life of various horticultural crops by inhibiting ethylene biosynthesis (Leshem et al 1998). Also, fumigation with 4 pp m NO attenuate s already induced senescence in Arabidopsis (Mishina et al ., 2007). PCD by Caspase activity Regulation of apoptosis in cells is regulated by nitrosative stress and is mediated by caspase activity (Kim et al., 2002) Nitrosative stress can p revent or induce apoptosis (Fisch et al., 2000; Kim et al., 1995; Kwon et al., 2001) Caspases are the family of cysteine proteases which can inhibit apoptotic signal cascade by nitrosylating thiols in catalytic site of caspase or promote apoptosis by
33 rele ase of Cytochrome c from mitochondria by initiating loss of mitochondrial membrane potential (Xu and Zhang, 2009) Higher concentrations of NO can react with peroxide to form ONOO and peroxide can cause mitochondrial damage including alteration in permeab ility transitions and thus resulting in Cytochrome c release and caspase activation leading to apoptosis via DNA fragmentation. Much effort has been directed at research to find if apoptosis causing caspases exist in plants. PCD by NO/ROS The burst of NO and H 2 O 2 under stress conditions and a balance between their concentrations triggers cell death (1 4 ) NO/H 2 O 2 ratio in the range of 0.25 2.0 induces cell death. Peroxynitrite ( ONOO ) is formed if balance is either in the favor of NO or O 2 ONOO is a po werful oxidant that can react with DNA leading to cellular damage and cytotoxicity (Radi, 2004) however, it is not an important intermediate in NO induced cell death It is also known to cause nitration of tyrosine residues in proteins and contribute to m embrane damage that occurs during sene scence (Wendehenne et al., 2001). (1 4 ) Fate/Removal of NO NO has been shown to be removed by oxygen to form nitrogen dioxide, which then rapidly degrades to nitrite and nitrate in aqueous solutions (Durner an d Klessig, 1999) ( 1 5 ) NO can also react with other potential signaling molecules that are likely to be produced simultaneously with NO ( Delledonne et al., 2001) One such chemical is the superoxide free radical (O 2 ) (Proyer and Squadrito, 1995). NO and superoxide
34 radicals together produce peroxynitrite radicals depending on the balance between NO and superoxides. Moreover, g lutathione and hemoglobin are considered prime targets of NO. NO binds with ferrous hemoglobin and give s rise to Hb NO complexes. The r eaction is reversible with the addition of reductants. Non symbiotic Hb from alfalfa, barley and Arabidopsis are known to react with NO resulting in its removal from solutions. G lutathione concentrations are 2 3 mM in plant cells (Ball et al., 2004), and formation of S glutathione (GSNO) could have an impact on concentration of free NO. GSNO is metabolized by S nitrosoglutathione reductase ( GSNOR ) which produces oxidized glutathione (GSSG). (1 5 ) Objective o f This T hesis The objective of this research was to determine the mechanism of CMNP in promoting abscission of sweet orange and from that mechanism determine if a mechanistic model can be developed that can be used as predictor for loosenin g by commercial industry as an aid to mechani cal harvesting. Chapter 2 explores the potential of NO as the signal that is produce by action of CMNP and moves to the
35 abscission zone to promote abscission. Chapter 3 re examines the role of ethylene as the signal that promotes abscission by CMNP. Alt hough progress had been made on the mechanism of action of CMNP, there is still not enough known to develop a mechanistic model to predict loosening that would be useful by the comme rcial industry. Therefore, in C hapter 4 an empirical model is described a nd partially validated with field data. Table 1 1. Some effects of exogenous applications of NO via SNP on plants. Species SNP concentrations Effect Citation Sunflower 0.1 mol Delay senescence Selcukcam and Cevahir,2008 Sweet orange 10 mol Cell deat h Saviani et al 2002 Arabidopsis 50 mol Cell death Clarke et al 2000 Soybean 500 mol No cell death Delledonne et al 1998
36 Figure 1 1. Physiological effects of CMNP in sweet orange.
37 CHAPTER 2 ROLE OF NITRIC OXIDE IN ABSCISSION OF SWEET ORA NGE Declining labor availability and rising harvest costs in citrus has led to research and development of harvesting machines as early as the 1960s Mechanization of citrus harvesting became a priority for the citrus industry to remain competitive in the global market. Currently, trunk and canopy shakers with or without frames have been employed in commercial groves in Florida and fruit removal efficiency varies from 70% to 95% (Whitney, 1999 and Brown, 2002). However, tree injury and the late season development of an abscission agent as an aid to mechanical harvesting was expected to decrease harvest cost by increasing removal rates, reduce shaking frequency of harvester resulting in less injury, and solving late season Valencia problem by the harvest window by 2 4 weeks ( Roka and B urns, 2007) Many abscission agents, such as, ethephon, cycloheximide, methy l jasmonate, coronatine, and CMNP have been tested in Florida citrus groves (Hartmond et al., 2000; Burns, 2002; Burns et al., 2003). Except CMNP, all other abscission agents have been rejected for various reasons, including causing severe leaf drop and ph ytotoxicity. Only CMNP met the above mentioned requirements to be used as an abscission agent in mechanical harvesting. An application for registration of CMNP as an abscission agent has been submitted to the EPA. The mod e of action of CMNP inside sweet o range is still unclear. The research performed on the mode of action of CMNP has shown that it creates anoxic conditions, reduces ATP formation, and up regulates gene expression involved in lipid signaling. CMNP inhibits alcohol dehydrogenase activity in c itrus peel tissue that leads to
38 acetaldehyde accumulation ( Alferez et al., 2005). CMNP, a nitro pyrazole, uncouples energetic membranes of chloroplast and mitochondria and also uncouples electrochemical potential of plasma membrane ( Alferez et al., 2005) w hich leads to activation of ATPase to restore membrane potential (Zharona and Vinogradov, 2004) that in turn depletes ATP content. CMNP may alter NAD linked production of ATP and both membrane linked and substrate level phosphorylation resulting in reduced ATP production ( Alferez et al., 2005). Further, CMNP increases activities of phospholipase A2 (PLA2) and lipoxyenases (LOX), and content of lipid hydroperoxide (LPO) increased in citrus peel tissue ( Alferez et al., 2005). G ene expression of lipoxygenase a nd phosphol ipase D i s upregulated in Arabidopsis le aves after CMNP treatment (Alferez et al., 2007). Phospholipase A2 (PLA2), lipoxyenases (LOX) are prime enzymes involved in the octadecanoid pathway indicating a link between lipid signaling and abscission in citrus upon CMNP application. Kender et al (2001) and Pozo et al (2004 ) concluded that abscission is independent of methyl jasmonate and ethylene in sweet orange. Despite the considerable research that has been done on CMNP, the mode of action of CMN P is not known. Previous research on nitro pyrazoles in mammals demonstrated that nitro substituted pyrazoles produce NO (Xuan and Chiou, 2003) and it produces NO to varying degrees depending upon the substitution of the nitro group on the pyrazole ring (X uan and Chiou, 2003). CMNP is also a nitro substituted pyrazole and its structure suggested release of NO or NO 2 Based on the evidence that nitro pyrazoles release NO in mammals, we hypothesized that CMNP produces NO and could be a signal in abscission s ignaling (2 1)
39 (2 1) NO plays diverse roles in higher plants which includes inhibiting oxidative stress enzymes activities (Clark et al 2000), cell death (Pedroso et al 2000; Saviani et al 2002), wound signaling (Orozco Cardenas and Ryan, 2002), and senescence (Leshem, 1996). In response to biotic and abiotic stress, plant s activate resistance responses by increasing levels of reactive oxygen species (ROS) and synthesis of salicylic acid (SA) (Durner et al., 1998; Durner and Klessig, 1999). Nitric oxide accumulates as a defense response in plant cells and plays a key role in disease resistance. NO regulated H 2 O 2 levels in tobacco plants by reversibly inhibiting catalase and ascorbate peroxidase (APX) enzymes (Clark e et al 2000) NO also induced h ypersensitive cell death response in soybean cultured cells via ROS and activated genes for synthesis of protective proteins independently (Delledonne et al., 1998). SNP induced cell death and DNA fragmentation in Taxus brevifolia haploid cultures at conce ntration of 0.1 mM (Pedroso et al., 2000) N O is reported to delay senescence and maturation ( Hu a n g and Kao, 2004). Fumigation of fruits with nitric oxide at 0.25 mol/L extended the post harvest life of crops, such as, strawberry, kiwi, broccoli, and cucu mber (Leshem et al., 1998). Wounding in leaves of Arabidopsis induced NO burst
40 and upregulated gene expression of allene oxide synthase (AOS), lipoxygenase (LOX), OPR3, key enzymes of octadecanoid pathway (Glazebrook, 2001; Huang et al., 2004). The followi ng study was conducted to test the hypothesis that NO would be produced in sufficient concentration by CMNP to promote abscission of sweet orange. Material and Methods Plant m ateri al and t reatments Citrus sinensis L. Osbeck) from the citrus grove at UF/IFAS, Immokalee were cut with a 3 inch stem attached and brought to the lab. Fruit were dipped in different concentrations of sodium nitroprusside (SNP) ranging from 1 to 5 mM for 15 minutes. Control fruit were treated w ith water. Fruit were left on the shelf to dry and loosely covered with plastic sheets to retain humidity. Four days after treatment, fruit detachment force was d etermined. There were 10 fruit per treatment. In a second experiment, eight Valencia trees we re randomly selected and four were sprayed with CMNP @ 300 ppm with Activator 90 to drip at a commercial grove (CPI ranch one, Immokalee, Fl). Peel samples were collected each day for NO, NO 2 and NO 3 assays. FDF was also measured each day for 4 days NO determination Nitric oxide was assayed using the hemoglobin method as described by Murphy and Noack (1992). This method is based on the reaction between the oxygenated ferrous form of hemoglobin (HbO 2 ) and NO, which produces the ferric form, methemoglobin (metHb), and nitrate (2 2) Hemoglobin Fe (II) O 2 + NO Hemoglobin Fe (III) + NO 3 ( 2 2 ) The application of reaction was used originally to show release of NO from nitrovasodilators ( Mayer and Beretta, 2008 ) T his technique has been used to measure generation of NO in tissues, cells, and extracts ( Murphy and Noack, 1992)
41 Preparation of oxyhemoglobin (HbO 2 ) stock solution Fresh preparation of HbO 2 made e very day is the crucial step with this method for accurate measurement of NO. It was p repared by dissolving 50 mg of hemoglobin in 50 mM potassium phosphate buffer (pH 7.4) and was further diluted to 30 ml by adding buffer to maximize yield of hemoglobin. Sodium hydrosulfite (15 mg) was added to the solution followed by continuously blowin g a light stream of O 2 into the flask. The resulting HbO 2 solution was then passed through Sephadex G 25 column for desalting. The purity and concentration of the resulting oxyhemoglobin solution was determ ined spectrophotometrically at 401 421 nm. The max imum absorbance peak at 415 nm indicates that HbO 2 is pure and the concentration was calculated using the Beer 131 mM 1 cm 1 St andard curve for NO measurement A nitric oxide releasing compound, SNP, was used t o calibrate the curve for accurate NO quantification. A series of SNP solutions of different concentrations ranging from 0.2 2.5 M were used to measure absorbance of metHb after reaction with 10 M HbO 2 and 0.1 mM cysteine as reductant in assay solution. Absorbance was measured spectrophotometrically at 421 nm. A linear relationship was found between SNP concentrations and NO production with r 2 of 0.99 Measurement of NO production The peel tissue (1200 mg) was ground in liquid nitrogen and extracted with 50 mM potassium phosphate buffer (pH 7.4), homogenized and centrifuged at 15000 rpm for 30 min. The collected supernatant was incubated with 10 M HbO 2 and 50 mM potassium phosphate buffer for 10 min in the
42 dark at room temperature. The absorbance was rec orded at 421 nm spectrophotometrically. NO evolution was expressed in nM/gm fresh weight. NO 2 determination Production of NO 2 in citrus flavedo samples was determined by the method described by Ding et al (1988) which was originally known as the Grie ss reagent method. The flavedo tissue was ground in liquid nitrogen and extracted with 50 mM glacial acetic acid buffer (pH 3.6), then homogenized and centrifuged at 15000 rpm for 30 min. The collected supernatant was incubated with 1% sulfanilamide and 0. 1% N 1 napthylethylenediamine dihydrochloride (NED) at room temperature for 10 min. The absorbance was determined in spectrophotometer at 550 nm. NO 2 evolution was expressed in nmol/g fresh wt. NO 3 determination The flavedo tissue (1200 mg) was ground in liquid nitrogen and extracted with 50 mM potassium phosphate buffer (pH 7.4). Nitrate content of the tissue sample was determined using a Flow Analyzer (Quinch Chem 8500, Lachat Co.) at 520 nm (Harbridge, 2007 a). Statistical analysis Standard error ( SE) was used to separate means for SNP treated fruit experiment. Differences among treatments was determined by T 0.05 (SAS Inst., Cary, NC) for the second experiment. Results and Discussion Fruit Detachment F orce of SNP and CMNP T r eated F ruits In order to verify that NO produced via CMNP could play a role in abscission, Valencia fruit were first treated with SNP, well known for releasing nitric oxide ( Bates et al., 1991 ). It was found that FDF was reduced by almost half with 3 or 5 mM SNP ( F igure 2 1 ) FDF of CMNP treated fruit declined by 50% compared to control fruit (Figure 2 2). S imilar results were found in various field studies conducted with CMNP at
43 2 mM where FDF declined by 50% three days after CMNP application ( E bel and Burns, 2008; Ebel et al., 2010 ; Kender et al., 2001; Yuan and Burns, 2004 ; Pozo et al., 2004; Burns et al., 2006). SNP has been shown to loosen fruit in a similar manner as CMNP. Considering the similarity between loosening pattern of SNP and CMNP, the data appears to indicate that NO could have a possible role in signaling of abscission. However, data was not collected on amount of NO produced from SNP inside the fruit and it was assumed that NO have been produced in enough amounts needed to cause loosening as supported by earlier studies (Clarke et al., 2000; Saviani et al., 2002). R esemblance in results of S NP and CMNP indicated that NO could be the signal travelling across the flavedo to the abscission zone triggering cascade of reactions leading to abscission. In addition, NO is known for its diverse roles in plants, such as, delaying senescence on the one hand and causing cell death on the other, depending on the concentration. Therefore, it was important to measure NO concentration produced in fruit peel tissue after CMNP application to determine if the amount was high enough to promote abscission NO, NO 2 and NO 3 C oncentrations after CMNP T reatment N itrite and nitrate ions are the primary end products of nitric oxide. To understand NO metabolism therefore measurement of NO 2 and NO 3 is necessary. NO (Figure 2 3) and NO 2 (Figure 2 4) content increase d slowly and peaked at day 2 and declined thereafter NO 3 concentration increased to d ay 3 and decreased thereafter (F igure 2 5). NO and NO 3 concentrations were in the nanomolar range while NO 2 concentrations were in the picomol ar range a fter CMNP app lication The results indicate that NO was converted to NO 2 which was then oxidized rapidly to its final product NO 3 T he total
44 amount of n itric oxide produced after CMNP application was in nanomolar range for this study. NO has growth promoting effe cts at low concentrations, and inhibitory effects at high concentrations (Beligini and Lamattina, 1999). NO delayed senescence at 0.1 M in sunflower (Selcukcam and Cavahir, 2008), in various fruits and vegetables at 0.25 M (Leshem et al 1998), and prom oted senescence in sunflower at 400 M (Selcukcam and Cavahir, 2008), and at 500 M in soybean (Delledonne et al., 1998, 2001). SNP induced programmed cell death in cultured sweet orange at 10 M concentration (Saviani et al 2002). In the present study, SNP at 3 mM and 5 mM concentration s appeared to produce NO in higher amounts in flavedo tissue to cause loosening. N ano molar concentrations of NO produced after CMNP application suggested a role more towards the protective side of metabolism in this stud y The normal physiological concentration of NO in plant tissues occur at pico to nano m olar ranges ( Planchet and Kaiser, 2006) whereas higher concentrations generally cause injury to plant tissues (Colasanti and Suzuki, 2000) Furthermore NO production is higher in young leaves than in older senescing ones (Magalhaes et al., 2000) indicating decrease in NO emissions that would promote maturation and senescence of plant organs I t was shown that NO and ROS are produced concomitantly in plants under stres s conditions ( Zhao, 2007 ) Depending on concentrations of NO and ROS, NO suppress es the toxic concentrations of ROS (Cheng et al., 2002), despite the synergistic effect of NO and ROS in PCD ( Delledonne et al 2001 ). NO at low concentration disrupts the cascade of reactions triggered by ROS and prevent injury (Durzan and Pedroso, 2002) and it enhances superoxide production in mitochondria at
45 higher concentration (Millar and Day, 1996) It was expected that plant s could undergo oxidative stress after CMNP application, therefore, production of hydrogen peroxide in flavedo was determined. Hydrogen peroxide produced in M concentration (Sharma and Ebel, unpublished) however no difference was found with the control (data not shown) which suggested no oxidative stress upon CMNP application. CMNP was shown to increase s uperoxide dismutase activity up to 5 days after CMNP application which would indicate increased production of H 2 O 2 (Kumar and Ebel, 2012) but it is apparent that H 2 O 2 catabolizing mechanisms mus t also be active to maintain H 2 O 2 concentrations similar to the control Thus, NO produced after CMNP application appears to promote protection of plant tissue from the damaging effects of ROS by upregulating activity of ROS scavenging enzymes. In summary, application of SNP to sweet orange indicated that NO in the millimolar range can promote abscission However, concentration of NO produced in flavedo tissue upon CMNP application was in the nanomolar range, which is too low to cause abscission. The data i ndicates that NO is involved in healing from the stress effects of CMNP.
46 Figure 2 1 and control fruit treated with water FDF data was expre ssed as means SE from 10 fruits per treatment. Fig ure 2 2 Fruit detachment force (FDF) the abscission agent CMNP and water (control) Means within each sampling time f ollowed by different letters indicate significant difference at P < 0.05.
47 Figure 2 3 treatment with the abscission agent CMNP. Means within each samp ling time followed by different letters indicate significant difference at P < 0.05. Figure 2 4 NO 2 treatment with abscission agent CMNP. Means within each sampling time followed by different letters indicate significant difference at P < 0.05
48 Figure 2 5 NO 3 concentration in flavedo tissue o treatment with abscission agent CMNP. Mean s within each sampling time followed by different letters indicate significant difference at P < 0.05.
49 CHAPTER 3 INVOLVEMENT OF ETHYLENE IN ABSCISSION OF SWEET ORANGE The mechanism by which CMNP loosens fruit is not known. The known parts of the meta bolic pathway of CMNP such as, lipid signaling, uncoupling ATPase, or anoxia like conditions did not appear to lead to abscission. Also, the alternative hypothesis that NO serves an abscission signal by CMNP application ha s been rejected due to NO producti on that is too low to promote loosening Ethylene is a naturally occurring gaseous hormone that plays a key role in regulating fruit ripening (Brady, 1987) and is an important promoter of abscission in higher plants (Abeles et al., 1992; Golding et al., 19 98). T here is the possibility that ethylene is the promoter of loosening by CMNP since its role has not been fully explored Ethylene is produced endogenously from methionine, producing S Adenosyl methionine (S AdoMet) via AdoMet synthetase, which is conve rted to 1 aminocyclopropane 1 carboxylate (ACC) catalyzed by ACC synthase (ACS), and subsequently oxidized to eth ylene by ACC oxidase ( 3 1). Binding of ethylene to receptors, such as, ETR 1, ETR 2, EIN 4, ERS 1 and ERS 2 (Hua et al., 1995, 1998; Sakai et a l., 1998) present in Arabidopsis membranes commences the signal transduction pathway. Ethylene biosynthetic pathway and steps of the pathway affected by the inhibitors AVG, STS and 1 MCP. (3 1)
50 The role of ethylene in climacteric fruit has been extensively studied over many years (Brady and Speirs, 1991). It is well known that there is an autocatalytic burst of ethy lene (system II) that initiates the ripening process (McMurchie et al., 1972). In non climacteric fruit, ethylene production is very low and inhibited by exogenous application of ethylene by autoinhibition (system I) (McMurchie et al., 1972). Goldschmidt ( 1997) divided non climacteric fruit into two categories. Fruit which do not respond to exogenous ethylene included cherry, grape, and strawberry (Pretel et al., 1995; Tian et al., 1997, 2000; Atta Aly et al., 2000). Fr uit, such as citrus are an exception since they lack a climacteric rise of ethylene, but exogenous ethylene accelerates respiration causing pigment changes in the peel (Purvis and Barmore, 1981; Stewart and Wheaton, 1972). Young detached citrus fruit demonstrate an autocatalytic rise in ethyl ene and increased respiration rates as they senesce during natural fruit drop shortly after flowering (Aharoni, 1968), a process Various studies have been conducted to determine the role of ethylene in citrus and o ther plant species by using ethylene inhibitors, such as, aminoethoxy vinyl glycine (AVG), silver thiosulfate (STS), and 1 methyl cyclopropene (1 MCP). AVG is a reversible inhibitor of ACC synthase (Boller et al., 1979), and thereby ethylene biosynthesis. Silver ions present in STS are believed to bind at the site of ethylene binding (Curtis 1981, 1982) and is reported to block ethylene induced abscission of leaves, fruits, and flowers (Beyer, 1976). 1 MCP is a cyclopropene, an ethylene binding or perceptio n inhibitor, similar in structure to ethylene ( Feng et al., 2004). STS at 0.5 mM decreased the autocatalytic production of ethylene in leaves and leaf discs of
51 concentrations (Riov and Yang, 1982). Incubation of grapefruit flavedo discs with AVG at various concentrations inhibited ethylene formation at lower concentrations but induced ACC synthase activity at higher concentrations (Mullins et al., 1999). As part of the research eff ort to determine the mode of action of 5 chloro 3 methyl 4 nitro 1H pyrazole (CMNP), a potential abscission agent used in sweet orange, Pozo et al (2004) conducted experiments to determine if ethylene served as an intermediate signal in abscission via CMN P by using 1 MCP. 1 MCP in combination with ethephon or CMNP prevented leaf drop, however, was not able to stop fruit Further, 1 MCP was unable to stop the reduction in fruit detachment force ( FDF ) in mechanically wounded Hamlin an d Valencia orange fruit (Kostenyuk and Burns, 2004 ) suggesting that wounding, ethephon and CMNP application may not require et hylene binding to its receptors, hence, this process may be ethylene independent. T he effect of any inhibitor depends upon the cr op, its state of development dos e of inhibitor, and the ability of the tissue to regenerate active sites for re gaining ethylene sensitivity (Blankenship and Dole, 2003). Stimulation of respiration in strawberry after 1 MCP treatment indicated that non cli macteric fruit, such as, strawberry may have different type s of ethylene receptors which may be 1 MCP independent (Tian et al., 2000). In the current study, AVG and STS were used to clarify the role of ethylene as a promoter of abscission of CMNP treated s weet orange. Materials and M ethods Plant m aterial s attached were collected from the citrus grove located at UF/IFAS, Immokalee and brought to the lab for conducting
52 the experiment. Cutting the fruit at the stem did not a lter fruit detachment force as shown in a previous stud y ( Sharma et al. 2012) Treatments AVG and STS were applied a t different concentrations in the first experiment to find the best concentration that retarded loosening by CMNP and without injuring th e fruit (Table 3 1). All fruit were dipped in CMNP, AVG and STS for one minute for treatment s 1 to 6. For treatments 7 11, f ruit were first treated with inhibitor for one minute and in C MNP thereafter for one minute Control fruit was dipped in water for 1 min. In the second experiment, f ruit were dipped in inhibitor solution and CMNP for 1 min each on day zero (Table 3 2). Inhibitor treatments ( 3x fruit were treated with AVG and STS after every 24 hours for 3 days while 1x treatments indicate that only one treatment of inhibitor was applied before CMNP treatement Fruit were treated for one minute in each inhibitor solution and with CMNP thereafter (4 through 7). Data Collected FDF measurement Three days after CMNP and in hibitor applications fruit detachment force was recorded with a Force One digital force gauge ( Wagner instruments, Greenwich, CT ) (Pozo et al., 2004) for both experiments. Ethylene measurement Ethyle ne was measured from fruit of exper iment 2. Fruit were enclosed in air tight jars ( 3.69 L) with a rubber stopper one hour prior to ethylene estima tion. Each treatment contained 4 jars having three fruit in each jar One syring e/ jar gave total ethylene for 3 fruit After one hour 1 ml of headspace from each jar was extracted using a 1 ml syringe and injected into a gas chromatograph (Model 5890A, Hewlett Packed, Wilmington, DE) fitted with a an activated alumina column
53 ( CTR1 column Alltech Assoc iates, Inc., Deerfield, IL) and flame ionization detector Heli um w as the carrier gas at a flow rate of 3.0 cm/s. Calculation of ethylene flux (E flux ) from the fruit surface To determine E flux (nanomol/cm 2 /s), the ethylene concentration from the headspace of the jars was converted to mol by first determining the et hylene concentration in ppm using E xc = [E sc x E xa )]/E sa ) where E xc was the ethylene concentration of the sample in ppm, E sc was the ethylene concentration of the standard (2.5 ppm), E xa was the area under the curve for the sample, and E sa was the area un der the curve for the standard. The ethylene concentration of the sample was converted to mol, assuming 1 ppm = 1 mg/L and using the equation: X nanomol = E xc (ppm) x 1 (mg/L)/1 (ppm) x 1 (g)/1000 (mg) x 1 (mol)/28.05 (g) x 1,000,000,000 nanomol/mol x [V jar (L) V fruit (L)] where the molecular weight of ethylene is 28.05 g and [V jar (L) V fruit (L)] is the volume in liters of the headspace in the jar minus the headspace lost by the presence of the fruit. The surface area of each fruit (cm 2 ) was determi ned by finding its displacement of water in a graduated cylinder and using the volume to find the radius of the fruit using the formula 3 ) and then using the radius and the formula for surface area of 2 ). The equation assumes that the fruit was a sphere which is a reasonable assu mption for sweet oranges. The time elapsed from the sealing of the jars to the removal of headspace with the syringe represented the time elapsed in seconds. Correlation between total ethylene produced and final fruit detachment force The total ethylene produced over the 3 days for each fruit was determined by integrating the area under E flux over time using the following equation (3 2) :
54 (3 2 ) w here E total is the total amount of ethylene produced per unit area by the fruit over 72 hours (n moles/cm 2 ), [(E flux 1 + E flux 2 )/2] is the average E flux of two consecutive measurements and t was the time elapsed between measurements. The correlation between E total and FDF was determined using the Proc REG procedure of SAS. Statistical analysis Data was analyzed as a CRD with 4 replications for ethylene and 12 replications for FDF. Results a nd Discussion FDF of Fruit Treated with AVG and STS in Preliminary and Second Experiment AVG and STS inhibit ethylene action at different concentrations in various horticultural crops. Since no specific concentration has been determined for citrus, two con centrations of AVG and three for STS were tested in the first experiment in order to Effect of CMNP and AVG on FDF In the first experiment (Figure 3 1 ), FDF of CMNP treated fr uit was half that of the control fruit which is similar to that found in many field studies Fruit treated with AVG (3x) at 10 M and 100 M alone did not loosen fruit such that FDF was similar to control. Fruit treated with AVG at 10 M (3x) and 100 M (3x) followed by CMNP were intermediate to the untreated controls and CMNP treated fruit. Since even the highest concentration of AVG did not prevent loosening by CMNP, 500 M was used in the second experiment to strongly inhibit loosening.
55 In the second e xperiment, FDF of CMNP treated fruit was about 50% than control fruit (Figure 3 2 ) which was similar to the first experiment Also similar to the first experiment, AVG (3x) at 500 M applied alone did n ot affect FDF which was similar to control fruit. FD F of fruit treated with AVG (3x) and AVG (1x) with CMNP was found to be intermediate between control and CMNP treated fruit. Both experiments demonstrated the same trend of loosening or not loosening fruit via AVG. However, in experiment 2, AVG at 500 M (3x) along with CMNP appeared to inhibit loosening such that FDF was closer to control fruit. Ethylene production and its e ffect on FDF Ethylene production in CMNP treated fruits increased gradually, peaking 72 hr after CMNP application whereas it was ne gligible i n cont rol fruit (F ig ure 3 3 ). Treatment of fruit with AVG (3x) produced little ethylene similar to the control and no loosening occurred as a result. E thylene production increased w hen AVG w as applied either only once (1x) or three times (3x) wit h CMNP as compared to control A little loosening was observed with AVG (1x) + CMNP treatment. E thylene production was 64% in AVG (3x) + CMNP treatment than the CMNP treated fruit Plant s under stress produce signals to generate ethylene (Abeles, 1973; Ya ng and Pratt, 1978). with 300 ppm CMNP was reported to have wounding like symptoms and promote d ethylene production that reache d a maximum b etween 3 to 4 days after application (Kossuth and Biggs, 1977). AVG inhibits ethylene p roduction via inhibiting that ACS enzyme in the biosynthetic pathway of ethylene. Application of AVG (3x) alone did not increase ethylene production compared to the control However, AVG plus CMNP promoted some ethylene production with the maximum occurrin g 72 hrs after fruit were t reated with AVG CMNP action promoted
56 ethylene production and applied concentration of AVG appeared not to be enough to block ethylene production. However, in this study, it appeared that AVG had been engaged more in blocking eth ylene produced by CMNP. A strong linear correlation (F ig ure 3 4 ) between FDF and total ethylene produced o ver the 72 hour period supports the hypothesis that the abscission of fruit is dependent on the amount of ethylene being produced and plays a n import ant part in abscission of sweet orange through CMNP application. CMNP produces anoxia like conditions inside fruit by inhibiting alcohol dehydrogenase (ADH) activity that leads to accumulation of acetaldehyde ( Alferez et al., 2005). Under flooding stres s, or hypoxic and anoxic conditions, ACC synthase activity increases and ACC accumulates in maize roots ( He et al., 1994, 1996 ) but is then transported to shoots which is then converted to ethylene ( Drew et al., 1979; Jackson et al., 1985; Atwell et al., 1 988 ), however, the presence of ethylene synthesis inhibitor s prevents formation of ethylene ( Drew, 1997 ; Drew et al., 2000 ). When CMNP was applied to peel in a band 1 cm below calyx or to any portion of peel, a signal moves from that section of peel to abs cission zone which activates the machinery responsible for dissolving middle lamella and subsequent abscission ( Sexton and Roberts, 1982) Due to anoxia like conditions created by CMNP, it could be conceived that ACC synthase becomes activated and ACC migh t accumulate in the CMNP treated flavedo, which then serves as the signal that moves to the abscission zone (AZ) where it i s converted to ethylene. The presence of AVG would prevent ACC synthase enzyme activity and inhibit ACC formation. ACC could be consi dered as one of the signal s travelling to the AZ where it is converted to ethylene as ACC is a polar molecule and is
57 easily soluble (Bradford and Yang, 1980) E thylene would serve as a poor signal from the pee l to the AZ since it is non polar molecule that is not readably soluble in water that makes it more prone to escape to atmosphere. Effect of CMNP and STS on FDF In the first experiment, FDF of CMNP treated fruit was considerably lower th an the control fruit ( F igure 3 1 ). All three concentrations of S TS (3x) did not loosen fruit and FDF was statistically similar to control. STS (3x) at all three concentrations (0.5, 10 and 50 mM) plus CMNP demonstrated a trend in which loosening was dependent on concentration of STS. STS at 0.5 mM plus CMNP caused loos ening to some extent and was intermediate between CMNP treated and control fruit. While, STS at 10 mM and 50 mM with CMNP did not loosen fruit and FDF was found higher than control fruit with these concentrations. A slight injury was noticed on fruit treat ed with 5 0 mM STS therefore, STS at 10 mM was used for second experiment. In the second experiment, CMNP abscise d the fruit the same way as it did in first experiment as compared to control ( Figure 3 2 ) Among all STS treatments, STS (3x) alone or with C MNP prevented loosening and FDF was statistically similar to the control. STS (1x) plus CMNP caused loosening similar to CMNP treated fruit and was not able to prevent abscission. Ethylene production and its e ffect on FDF When fruit were treated with STS alone or with CMNP, ethylene rose within the first 24 hr and peaked at 36 hr for all STS treatments, however, it declined with time thereafter (F igure 3 5 ). The most ethylene was produced with STS (1x) + CMNP followed by STS (3x) + CMNP and STS (3x). Ethyl ene produced by STS (3x) did not cause abscission like it prevented in the first experiment. STS (3x) + CMNP prevented
58 abscission despite the amount of ethylene evolved in this treatment and was similar to th e FDF found in the first experiment. FDF of frui t treated with STS (1x) + CMNP showed a huge decline as compared to control and was statistic ally similar to CMNP (Figure 3 2 ) which showed that multiple applications of STS (3x) was necessary to stop abscission with CMNP A single application of STS (1x) with CMNP was not enough to block ethy lene binding sites which may be regenerating. Treatment of STS after 4 hours of ethylene application progressively reduced its effectiveness and inhibited petiole abscission in Coleus explants ( Baird et al., 1984). I n this study, fruit were given STS treatment after every 12 hours which possibly provided enough time to regenerate receptors. E thylene produced as a result of STS application appeared to concentration dependent S ilver nitrate concentrations between 1 to 100 molar w ere reported to reduce ethylene production and delay senescence and abscission in bean explants (Kushad and Poovaiah, 1984). Further, STS retarded ethylene induced abscission of leaves and berries of English holly and American Mistletoe at a co ncentration of 1 mol of Ag + and stimulated leaf abscission at high 4 mol of Ag + (Joyce et al., 1990). The concentration of STS used in this experiment was comparatively high which could be one of the reasons that resulted in excessive ethylene evolution in response to stress posed by STS along with CMNP However, application of 4 mM STS increased ethylene formation in Poinsettia brackets but prevented epinasty (Reid et al., 1981). Further, 10 ppm Ag + alone or in combination of CO 2 increased ethylene produ ction in the first 18 hours after application and prevented leaf senescence (Aharoni and Lieberman, 1979). Also, application of STS before ethylene treatment reduce d activities of cellulases and
59 polygalacturonase in the abscission zone 50% and 68% respectively 24 48 hours a f ter ethylene treatment (Brown and Burns, 1998). Biotic and abiotic stress conditions are also shown to cause fruit abscission (Aloni et al., 1991; Ebel et al., 2001). Due to the promotion of ethylene evolution by STS as well as its inhibition in ethylene binding, its not surprising that the correlation between total ethylene produced and FDF was not high (Figure 3 6 ). Apart from preventing abscission, it appeared that STS had additional effects on plant metabolism apar t from regulating abscission, which, as a result, did not produce a strong correlation between FDF and ethylene produced via STS and CMNP. On the whole, results from both experiments demonstrated that STS hindered the abscission process by blocking ethylen e binding sites which clearly indicated that abscission via CMNP is an ethylene dependent process which requires binding of ethylene to the active site for abscission to occur. Both STS and 1 MCP are known as ethylene binding site inhibitors, however, the y inhibit the binding site differently Veen (1986) h ypoth esized that the ethylene binding site consist s of two subunits, a proteinaceous regulatory subunit A, and an enzyme subunit B with a copper atom. Binding of ethylene to subunit A causes allosteric c hanges to the protein structure and then it binds to subunit B which causes ethylene action STS inhibit s ethylene action by replac ing the copper ion in subunit B with a silver ion resulting in no binding to subunit A and thus prevents a response 1 MCP w hich also inhibits the binding site of the ethylene receptor promoting abscission in sweet orange and thus the authors concluded that there must be an ethylene independent pathway by which CMNP promotes abscission (P ozo e t al.,
60 2004; Kostenyuk and Burns, 2004 ) One possible reason for the non responsiveness of 1 MCP in their studies could be due to its high volatility and hence, its inability to get into the fruit in high enough concentration to prevent abscission after s pray in the field (Agro Fresh, personal communication) Another possible explanation for the lack of a response of sweet orange abscission zones to 1 MCP may be due to an ethylene binding site that is too small to accommodate 1 MCP Ethylene (a) propylen e (b) and 1 MCP (c) have structural simil arities, however, that latter two are larger than ethylene itself. Furthermore, propylene which has been shown in many studies to produce ethylene like effects, is much less active than ethylene itself, presumabl y because of its greater difficulty in binding to the ethylene receptor binding site ( Pratt and Goeschl, 1969) 1 MCP is even larger than propylene and thus may have even greater difficulty in binding to the active site of the ethylene receptor in sweet o range. Such a case would lead to the erroneous conclusion that independent. a) Ethylene b) Propylene c) 1 MCP
61 The results of this study support s the hypothesis that CMNP promoted production of ethylene and that ethylene was involved in abscission of sweet orange. Use of AVG inhibited ethylene production and caused no loosening demonstrating a role of ethylene in abscission. However, CMNP can evoke abscission up to 1 cm from the abscission zone An alternative hypothesis is t hat ACC, a precursor of ethylene biosynthesis, is the most likely signal moving from peel to the abscission zone as described earlier. The r esponse of abscission to AVG and STS but not to 1 MCP indicated that the ethylene bi nding recep tor in s weet orange is 1 MCP independent. Based on the current study and data that has been published in the literature, we proposed the following mechanistic model by which CMNP prom otes loosening through ethylene (3 3). The model contains step s from uptake of CMNP to loosening. Uptake of CMNP was reported to cause stress, such as, anoxia like conditions by inhibiting alcohol dehydrogenase (ADH) activity in the flavedo tissue (Alferez et al., 2007). Under this kind of stress, plant tissue s accum ulate ACC in higher amounts by increasing ACS transcripts (Yang and Hoffman, 1984; Bleecker and Kende, 2000). There is evidence that ACC accumulates in waterlogged roots of maize and tomato plant (Bradford and Yang, 1980) w hich is transported up in the ste m to the adjacent cells where it can convert to ethylene by ACO. Similarly, it can be hypothesized that ACC accumulate s in peel tissue after CMNP application under anoxi c conditions which would travel through the peel to the abscission zone where it is con verted to ethylene
62 (3 3) Table 3 1. Treatment of fruit with AVG, STS and CMNP in the first experiment Treatments 1 2 mM CMNP 2 10 M AVG 3 100 M AVG 4 0.5 mM STS 5 10 mM STS 6 50 mM STS 7 2mM CMNP + 10 M AVG 8 2 mM CMNP + 100 M AVG 9 2 mM C MNP + 0.5 mM STS 10 2 mM CMNP + 10 mM STS 11 2 mM CMNP + 50 mM STS 12 Water (Control)
63 Table 3 2. Treatment of fruit with AVG, STS and CMNP in second experiment. Treatments 1 2 mM CMNP 2 500 M AVG 3 10 mM STS 4 500 M AVG (3x) + 2 mM CMNP 5 10 mM STS (3x) + 2 mM CMNP 6 500 M AVG (1x) + 2 mM CMNP 7 10 mM STS (1x) + 2 mM CMNP 8 Water (control)
64 Figure 3 1 Fruit detachment force of fruit treated with CMNP at 2 mM and inhibitors at different conc entrations in preliminary experiment. Difference in means was
65 Figure 3 2 Fruit detachment force of fruit treated with CMNP at 2 mM and inhibitors at different concentratio ns in second experiment. Difference in means was Figure 3 3 Ethylene the abscission agent CMNP and the ethy lene synthesis inhibitor AVG Ethylene data was expressed as means SE from 12 fruits per treatment Bars not visible lie within the height of the data symbol.
66 Figure 3 4 Fruit detachment force (FDF) correlated wi th the total ethylene flux over the 72 hour period after CMNP treatment. significant to the P = 0.02 level. Figure 3 5 ment with abscission agent CMNP and STS. Ethylene data was expressed as means SE from 12 fruits per treatment.
6 7 Figure 3 6 Fruit detachment force (FDF) correlated with the total ethylene flux over the 72 hour period after CMNP treatment. significant to the P = 0.35 level.
68 CHAPTER 4 A PREDICTIVE MODEL FOR PROMOTING ABSCISSION OF SWEET ORANGES AT VARYING TEMPERATURES BY AN ABSCISSION AGENT There are 193, 000 ha sweet orange grown commercially for the juice industry in Florida, but only 7% of the area is mechanically harvested ( Anonymous 2008). Despite the low proportion of the industry being mechanically harvested, there has been great interest to expand the acreage. The most co mmonly used mechanical harvesting systems are canopy shakers which harvest fruit by vibrating mechanism of tines that impact fruit directly or fruit bearing branches (Whitney and Hedden, 1973; Ebel et al., 2009). Two types of canopy shakers are currently u sed to harvest sweet oranges. Pull behind canopy shakers are pulled behind a tractor and leave harvested fruit on the ground that are picked up by laborers (Whitney, 1999; Bora et al., 2006). Self propelled canopy shakers are equipped with decks positioned under the tree to catch the fruit during shaking. This type of shaker works in pairs on opposite sides of a tree row. Harvesting efficiency of currently employed mechanical harvesters is not consistent and recovers from 70% to 85% of the total available c rop. The rest of the fruit on the ground or left in the tree is gleaned from the grove by manual labor. To promote removal efficiency by mechanical harvesters, research has been conducted on agents that would promote abscission (Whitney et al., 2000a, 200 0b, 2001). Of all abscission agents studied so far, 5 chloro 3 methyl 4 nitro 1H pyrazole (CMNP) has been shown to be the most promising (Wilson, 1973; Whitney, 1975, 1976; Freeman and Sarooshi, 1976) such that an application for its registration has been submitted to the EPA. Efficacy of CMNP has been shown to be affected by many factors such as, concentration, coverage, post spray precipitation and air temperature (Kender and Hartmond, 1999; BenSalem et al., 2001; Salyani et al., 2002; Burns et al.,
69 2006; Ebel and Burns, 2008). Of all factors, temperature has the most significant impact on efficacy of CMNP. Maximum uptake of CMNP occurs at 25 to 35 o C with very little getting into the peel 10 o C (Murphy et al., 1976). Once inside the fruit, loosening by CMN P has also been shown to be temperature dependent. The rate of loosening by CMNP begins to decline when temperatures decline below about 20 o C and essentially stops when temperatures decline below 15 o C (Yuan and Burns, 2004). These temperatures typically occur throughout the winter harvesting months in Florida (Barkatay et al., 2011). Cold temperatures slow the rate of loosening which would affect scheduling of CMNP applications and harvest. The commercial sweet orange industry in Florida would be aided b y a mathematical model that predicts the rate of loosening. The ideal model would be based on the mode of action of CMNP, however, the mode of action is still unknown. The objective of this study was to develop an empirical model that fits data from seve ral trials that determined the rate of loosening of sweet orange by CMNP juice industry. Material and Methods Theoretical development of the m odel The model was develo ped in 4 steps. Firs t, l environmental growth chamber at various temperatures as published by Yuan et al., (2004). The trees were sprayed with 200 mM CMNP and FDF was measured 120 hr after application. The equation was of the form: FDF m(N) = (4 1)
70 where FDF m(N) is an estimation of the FDF with units in Newtons. FDF min represents the minimum FDF reached after 120 hr after CMNP application and represents the lower asymptote. FDF t=0 is the FDF immediately before CMNP was applied and represents the upper asymptote. CMNP has been shown in several studies to consistently loosen fruit to about 10 before CMNP application ( Ebel and Burns, 2008 ). T is air temperature and T inf is the inflection point of the curve which was 18 o C. The only coefficient in the regression that had to be determined stat procedure of the S tatistical Analysis System (Figure 4 1). Second, Eq. (4 1) was converted to predict the hourly rate of loosening (FDF p(N/hr) ) by dividing by the amount of time that field studies have shown is the effect loosening period at optimal temper atures, which is about 48 hours. FDF p(N/hr) = (4 2) Third, the model was adjusted by including a quantity that would adjust the rate of loosening based on the concentration of CMNP applied. The decline in FDF by CMNP at any given temperature has been shown previously to be linearly related for CMNP concentrations in the range of 0 to 300 ppm with the y intercept at the origin ( Burns et al., 2005). Since Eq uations (4 1 ) and (4 2 ) were developed based on CM NP applied at 200 ppm, the adjustment would take the form: FDF p(N/hr) = (4 3) where C CMNP(ppm) is the concentration of CMNP applied in parts per million.
71 Fourth, the predicted FDF at each hour (FDF pt(N) ) after CMNP application was determined by the accumulation of the calculated hourly reduction in FDF from t=0 to t: FDF pt(N) = (4 4) Mod el v alidation The model was validated by spraying fully mature trees in a commercial grove with CMNP, measuring air temperature and comparing the actual Citrus paradisi Poncirus trifoliata ) and Carrizo Citrange ( Citrus sinensis [L.] Osb Poncirus trifoliata [L.] Raf.) 3.4 m in Ft. Drum sa nd (siliceous, hyperthermic Aeric Endoaquepts) and Malabar fine sand (siliceous, hyperthermic Grossarenic Endoaqualfs) located at Silver Strand North grove, were spaced 6. 7 3.7 m and grown on a Wabasso fine sandy soil (siliceous, active, hyperthermic Alfic Alaquods) located at Ranch One Cooperative Inc., block P26 (Lat. 26 CMNP (ASI 100 17 EC, 17.2% w/w) was applied at 200 and 300 ppm with 0.1% (w/v) Activator 90 (alkylphenol ethoxylate, alcohol ethoxylate, and oil fatty acid; no. 268173B, Northern Tool and Equipment Co., Faribault, MN). Three tri als were conducted for 11, Mar. 30, Apr. 6 and Apr. 10). At each trial, four trees per CMNP treatment in a ran domized complete
72 block design (RCBD) were sprayed. There was at least one buffer tree between treated trees. Air temperature was measured hourly using a portable weather station that was located between two trees in the center of the block and at 1.5 m he ight. Once or twice a day starting immediately before CMNP application to 5 days after CMNP application, 10 fruit were randomly clipped from each tree at the stem and FDF measured immediately using a force gauge (Force One digital force gauge; Wagner Instr uments, Greenwich, CT) as described previously (Pozo et al., 2004). Results and Discussion Model Development The model was developed using generalized logistic function because of it being confined by upper and lower asymptotes, which has been shown in a previous study (Yuan and Burns, 2004). The upper and lower asymptotes and inflection point (18 o C) constants were estimated from the original data by Yuan and Burns (2004). The logistic function fit the data with a regress ion coefficient near unity (Figu re 4 1). The only linear procedure of the Statistical Analysis System. Despite the excellent fit of the logistic function, some constants used in the model may not be satisfactory in predict ing loosening under other conditions. The upper asymptote value is based on the values from the original publication (Yuan and Burns, 2004), however, it is to be expected that the upper value will vary within the commercial industry. For example, it is k most years as it reaches its maximum maturation ( Ismail, 1971; Ebel et al., 2010 ).
73 characterized by a reduction in loos ening by CMNP due to internal hormonal factors ( Yuan et al., 2001, 2003). Furthermore, HLB is known to affect the FDF of sweet orange (Burns et al ., 2011 ) and with it being widespread throughout the industry ( Burrow et al., 2008) it is not known how it ma y affect predictability of the model. Estimating the lower asymptote is a particular problem because of the extremely high variability of FDF within the tree (Sharma et al., 2012). Sharma et al (2012) showed that FDF can vary as much as 5 120 N within the same tree of fully mature within the tree loosened uniformly such that after 4 days fruit began to drop. The fruit would drop as the force for detachment declined at or below the weight of the fruit. Dropped fruit are not used when measuring FDF thus as drop accelerates the lower FDF values become skewed. Thus, the lower asymptote value as shown in Figure ( 4 1 ) may be slightly high. Nevertheless, it is possible a nd would simplify use of the model to set the minimum FDF value at some percentage of the maximum value. For this model, FDF min 20% of FDF max and 40 50% of FDF max for th day after CMNP application. Thes e values are based on several field studies that have been conducted on FDF of these two cultivars after CMNP application (Kender et al., 2001; Burns, 2002; Pozo et al., 2004). In scheduling CMNP application and harvest under the threat of low air temperat ures, the timing of harvest would be delayed but can be manipulated by the concentration of CMNP applied The data used to develop Figure (4 1) was based on decline linearly f
74 concentration data on any given day ( Burns et al., 2005 ). Thus, multiplying the calculation of rate of decline of FDF by the concentration of CMNP divided by 200 ppm as shown in Eq. 4 3 is reasonable. As shown in a previous study ( Burns et al., 2005), however, the upper concentration that can be applied should be limited to 300 ppm since higher concentrations did not provide additional loosening. It should be noted that all applicati ons require complete coverage (to drip) since CMNP must come into direct contact with the peel such that only the concentration of CMNP can be manipulated and not the volume of spray applied on a per acre basis. The optimum volume of application for full coverage of spray should be 300 gal/acre (Ebel et al., 2009). With this volume, the maximum concentration will be allowed will be 300 ppm, based on the maximum a.i./acre that will be allowed under the label (Agrosource, personal communication). Model Vali dation The model was tested in a commercial grove by measuring FDF after CMNP application and comparing the measured values against those predicted by the model in equation 4 4 (Fig ure 4 clos C MNP application before it would decline, although by 3 rd day the measured FDF were close to the predicted value. The pattern for 300 ppm was not the same for 200 ppm data so there may be a difference in cultivar response to CMNP. The delay in
75 studies ( Kossuth and Biggs., 1977; Burns, 2002 ). The measured and predicted FDF also diverged after 3 days after application for values actually increased. Part of the reason for this as discussed earlier was due to fruit drop, which began 3 days after application an d accelerated up to 5 days after application (data not shown). Fruit that dropped from the tree were not used to determine FDF thus the average FDF would be skewed higher. Furthermore, the healing effects of nitric oxide (Chapter 2) could possible cause a reverse of abscission, although this needs to be confirmed. Nevertheless, other field studies have et al., 2004; Burns, 2002; Ebel et al., 2010 ). Temperatures often decline below 20 o C, below which the rate of loosening declines, during winter in Florida when sweet oranges are harvested (Barkatay et al., 2011). Thus it would be expected that scheduling of CMNP application and harvest in commercial groves will be affected by air temperature. This will be especially true for are harvested from late March into early June The model is shown to predict below 20 o C, especially at night throughout the loosening period (Fig ure 4 3). For both dates, even day time temperatures were below the 20 o C critical maximum below which loosening declined. For the Jan. 12 th application date, temperatures were cold initially
76 even declining below freezing the first night. For the Dec. 19 th application date, daytime temperatures limited loosening on the th ird day after CMNP application. As expected, the predicted rate of decline slowed during these cold periods, which was also reflected in measured FDF. by the abscission agent CMNP during cold temperatures. The model was based on previously published studies and tested against CMNP applications on several dates that included cold temperatures and provided a reasonable prediction compared to measured val ues. The grove in which the model was tested was under a single set of conditions, therefore the model requires further testing in a wider range of conditions that exist throughout the sweet orange industry in Florida where CMNP may be used.
77 Figure 4 1. Goodness of fit of a logistic function to fruit detachment force as affected by air temperature. The function was fitted to data of Yuan and Burns (2004).
78 Figure 4 2. Average measured and predicted fruit detachment force treatment and 10 fruit per tree, thus each data point represents 120 fruit for determined using equation 4 4 in the text. Measured FDF symbols not shown are hidden underneath the symbols for the predicted values. The vertical bars indicate 2X SE of the mean for measured values.
79 Figure 4 dates during the coldest parts of the winter months when this cultivar is normally harvested in Flor ida. The open circles in the bottom graphs represent measured fruit detachment force (FDF) and the closed circles the predicted FDF determined from equation 4 4 in the text.
80 CHAPTER 5 FUTURE RESEARCH AND SUGGESTIONS Mode of A ction In this study, ni tric oxide has been shown to be involved in the healing mechanism and the results support the hypothesis that ethylene i s involved in abscission. However, the entire mode of action of CMNP is still unclear. A mechanistic model was proposed in chapter 3 for CMNP action in which ACC has been suggested as a signaling molecule travelling from peel tissue to the abscission zone. Five steps in the model have been identified that could be temperature dependent including uptake of CMNP, ACC synthase (ACS) activity ACC movement from peel to AZ, up regulation of cell wall enzymes, and activity of cell wall enzymes. Of all these steps, ACS activity had been shown to be affected by low temperature in various studies (Zacarias et al., 2003; Field and Barrowclough, 1988 ) with the temperature curve for ACC synthase enzyme activity being similar to for temperature range for ethylene production (Borochov et al., 1985). In addition, the ACC synthase temperature curve resembled the FDF versus temperature curve upon applicatio n of CMNP which indicate s that ACC synthase may be the temperature lim iting step in the mechanism. However, accumulation of ACC upon CMNP application and decreased activity of ACS at low temperature in sweet orange would require further investigation. Apa rt from involvement of ethylene, CMNP could have its impact on r atio of auxin to ABA Activity of peroxidases increased in first 48 hours after CMNP application (Kumar and Ebel, 2012) which might be resulting in catabolism of auxins and decline in its cont ent. ABA has been shown to accumulate in roots of Cleopatra mandarin along
81 with ACC under water stress (Gomez Cadenas et al., 1996) Therefore content of ABA could increase after CMNP application. Mystery of selective abscission by CMNP needs to be resolv ed which could answer a lot of questions about the signaling mechanism promoting abscission. Empirical Model The empirical model that predicts FDF after CMNP application but del for Valencia more steps need to be included in the model other than temperature that could explain the wide range of variation in the FDF data. A n empirical mode of action of CMNP is figured out along with the rate limiting step most affected by temperature. Fruit drop appeared to be a major limitation to this model that needs to be addressed to increase predictability of the model More attention is required towards application of the model to greening infect ed trees because greening decreases FDF by 10 15% Also, m odel needs further validation with different sets of data at different locations.
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100 BIOGRAPHICAL SKETCH Sunehali Sharma was born in India. She completed her high school from Government Girls Senior Secondary School, Bathi nda (Punjab). She received her Bac helor of Science and Master of S cience from Punjab Agricultural University (PAU), Ludhiana, India. She joined the doctoral program at University of Florida in August 2009 in Horticultural Sciences department and graduated in December 2013.