FRUIT DETACHMENT IN BLUEBERRY by TRIPTI VASHISTH (Under the Direction of Anish Malladi) ABSTRACT A better understanding of fruit detachment and the processes mediating it is essential to improve the efficiency of mechanical harvesting in blueberry (Vaccinium sp.). In blueberry, fruit detachment may occur either at the point of attachment of the pedicel to the peduncle (peduncle- pedicel junction, PPJ) or at the point of attachment of the pedicel to the fruit (fruit-pedicel junction, FPJ). Whether fruit detachment at these junctions is mediated by the physiological process of abscission or through physical separation of the organ from the parent plant is not well understood. Abscission is a physiological process that involves the programmed separation of entire organs at an anatomically distinct layer called the abscission zone (AZ). Additionally, the spatial and temporal changes in the cell wall composition and metabolism and the regulation of fruit detachment process is not completely understood. In this study, a series of experiments were performed to understand physiological, biochemical and molecular aspects of fruit detachment in blueberry. Anatomical, physiological and microscopic analysis revealed that PPJ is the true abscission zone of blueberry and detachment at FPJ is as result of physical breakage. Glycome profiling and immuno-localization of PPJ indicated that alteration in pectins and hemicellulose plays a key role in cell separation during abscission. A number of cell-wall carbohydrate metabolism related genes were altered upon induction of abscission. RNA-Seq analysis showed that abscission agent induced abscission was associated with extensive changes in the expression of genes associated with the biosynthesis and signaling of phytohormones such as ethylene, jasmonic acid and auxin. Also, potentially a plant hormone cross-talk and interaction plays an important role in abscission. Scanning electron microscopy analysis and fruit detachment in response to mechanical shaking of southern highbush cultivar 'Suziblue' and accession line TH729 revealed that variability in ease of fruit detachment is due to stronger PPJ. A decrease in expression of cell wall hydrolysis related gene was observed with stronger PPJ. Potentially these phytohormone and cell wall hydrolysis genes play an important role in the genetic ease of fruit detachment/abscission. INDEX WORDS: blueberry, abscission, fruit detachment, MeJa, ethephon, glycome profiling, immuno-localization, RNA-Seq FRUIT DETACHMENT IN BLUEBERRY by TRIPTI VASHISTH B.Tech., Bundelkhand University, India, 2006 M.S., University of Georgia, 2009 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2013 © 2013 Tripti Vashisth All Rights Reserved FRUIT DETACHMENT IN BLUEBERRY by TRIPTI VASHISTH Major Professor: Anish Malladi Committee: Dayton Wilde Rakesh Singh Robert Shewfelt Scott NeSmith Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia December 2013 DEDICATION I would like to dedicate all my work to my parents Mr. OP Sharma and Mrs. Urmila Sharma and my husband Mr. Vijendra Sharma. Without their love and support, I would never have been able to accomplish this. iv ACKNOWLEDGEMENTS I would like to thank Dr. Anish Malladi for giving me the opportunity to work and learn under his guidance. Thank you Dr. Malladi for your support, guidance, listening to me and always showing me the right direction. You have been always there to correct me, and helped in learning, to do the things in a remarkable way. I would also like to thank Dr. NeSmith, Dr. Shewfelt, Dr. Singh, and Dr. Wilde, for serving as my committee members. I am also thankful to whole faculty and staff of Horticulture Department for their support throughout my research. Special thanks to Lisa Johnson and Madhumita Dash for being supportive and helping me throughout. I would also like to thank Justin Porter, Kristin Abney, Jim Gegogeine and all the student in Horticulture Department for helping me out specially in cutting abscission zone. I want to especially thank my father and mother, and my husband for always supporting me, believing in me and being a light during dark phases of my life. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS.............................................................................................................v CHAPTER 1 INTRODUCTION AND REVIEW OF LITERATURE ...............................................1 2 FRUIT DETACHMENT IN RABBITEYE BLUEBERRY: ABSCISSION OR PHYSICAL SEPARATION ..................................................................................16 3 CHANGES IN THE CELL WALL COMPOSITION AND WALL-RELATED GENE EXPRESSION DURING BLUEBERRY FRUIT ABSCISSION .............42 4 TRANSCRIPTOME WIDE CHANGES DURING THE INDUCTION OF ABSCISSION IN BLUEBERRY FRUIT..............................................................85 5 VARIABILITY IN THE EASE-OF-FRUIT DETACHMENT IN BLUEBERRY...125 6 CONCLUSION..........................................................................................................148 vi CHAPTER 1 INTRODUCTION AND REVIEW OF LITERATURE Blueberry (Vaccinium sps.) is an indigenous crop of the United States. Blueberry is a flowering plant with berries ranging from purple to black in color. North America is the largest producer contributing approximately 90% of the world’s blueberries. The most widely grown species of blueberry are northern highbush, southern highbush, and rabbiteye. In 2009, the total production of blueberries was around 207 million kilograms, which was worth over 500 million U.S. dollars (USDA). According to the USDA, consumption of fresh blueberries in 2007 was 0.26 kg per person, while consumption of frozen blueberries was an additional 0.14 kg per person (www.agmrc.org). The demand and market of fresh blueberries has increased tremendously in last few decades. Therefore, continuous efforts are being made to improve production and other aspects such as fruit quality in blueberry. Hand harvesting of rabbiteye and highbush blueberries is labor intensive and requires as many as 520 h of labor per acre (Brown et al., 1996). Hand harvesting costs range from $1.10 to 1.54 per kg for southern highbush blueberries and $0.86 to 1.10 per kg for rabbiteye blueberries (Safley et al., 2005). Harvesting of blueberries is the largest expense in the production of the blueberry crop (Yarborough, 1992). Hence there is a strong interest in reducing harvesting costs using mechanical harvesting. Mechanical harvesting can potentially reduce costs to about $0.26 per kg for rabbiteye blueberry. Mechanical harvesting of blueberries has been done commercially since 1966 (Austin and Williamson 1977, Mainland et al. 1975). Mechanical harvesters are typically over the row 1 harvesters and detach the berries from the bush through physical shaking of the bush. It was reported by Austin and Williamson (1977) that a greater amount of ripe rabbiteye blueberries were lost on the ground compared to harvested berries as a result of mechanical harvesting. Some green and unripe fruits were also removed by mechanical harvesting, resulting in additional losses. Sorting and grading of machine-harvested fruit on a commercial cleaning line further soften the berries resulting in greater decay in storage (Mainland et al. 1971, Mainland et al. 1975). Excessive vibration in mechanical harvesting operations often results in breakage of the pedicel, or detachment of the fruit away from the pedicel-berry junction. Therefore, such mechanical harvesting methods can ultimately lead to fruit injury and loss of overall fruit quality (Howell et al., 1976). Hence, methods to increase the mechanical harvesting efficiency are required. Abscission Abscission is a physiological process that involves the programmed separation of entire organs, such as leaves, petals, fruit, and flowers. Abscission is a highly coordinated and regulated process in response to plant developmental cues, and various biotic and abiotic stresses. Organ separation occurs at abscission zones (AZ). Abscission involves the breakdown of cell walls. The spatial and temporal regulation of the dissolution of primary cell wall polysaccharides and middle lamella is not completely understood, but many studies have indicated that the loss of pectins and other polysaccharides from the middle lamella and the primary cell wall is associated with abscission (Clements and Atkins, 2001; Roberts et al., 2000; Uheda and Nakamura, 2000). Fruit abscission in blueberry can occur either at the peduncle-pedicel junction (branch AZ) or the pedicel-berry junction (fruit AZ) (Gough and Litke, 1980). Enhancing the abscission characteristics of blueberry fruit can lead to increased efficiency of mechanical harvesting. 2 Abscission can be divided into four major steps (Patterson, 2007). During the first step formation of the AZ occurs. In step 2, AZ cells become responsive to phytohormones such as ethylene (by far the most extensively studied), jasmonates, auxin and abscissic acid (ABA). In response to the phytohormone signaling the middle lamella is dissolved. During step 3, cells in the AZ expand and cell wall loosening occurs. Finally in step 4, cell separation occurs followed by the suberization of the proximal layer of cells to form the protective layer. 1. Formation of abscission zone The abscission zone is comprised of several layers of small, densely cytoplasmic cells at the point of