‘lHull“NW!WIWMWlWllHlWllHl WI” 01—) 01-F- 01 TH "-0. .1 .1 Q, 4 {:13 I‘s A} 3 {FN/ LIBRARY Michigan State University This is to certify that the thesis entitled MICROARRAY ANALYSIS OF RICE GRAIN ABSCISSION REGULATED BY 8H4 presented by Ailing Zhou has been accepted towards fulfillment of the requirements for the MS. degree in Plant Biology Major Profe sor’s Signature 107/! /07 Date MSU is an affirmative-action, equal-opportunity employer PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DAIEDUE DAIEDUE DATEDUE 6/07 p:/ClRC/DateDue.indd-p.1 MICROARRAY ANALYSIS OF RICE GRAIN ABSCISSION REGULATED BY 8H4 By Ailing Zhou ATHESIS Submitted to Michigan State University In partial fulfillment of the requirements For the degree of MASTER OF SCIENCE Department of Plant Biology 2007 ABSTRACT MICROARRAY ANALYSIS OF RICE GRAIN ABSCISSION REGULATED BY 8H4 By Ailing Zhou Abscission is the programmed organ separation from the main body of the mother plant. Separation takes place at predetermined positions called abscission zones. Although abscission is essential for the plant life cycle, the molecular regulation of abscission remains poorly understood. The rice grain shattering gene sh4, which encodes a putative MYBB transcription factor, is involved in the development and function of the abscission zone between a rice grain and its pedicel. To investigate genes potentially regulated by SM during the abscission process, microarray analysis was conducted using sh4 transgenic plants. The study identified several categories of genes that were up-regulated by SM. These include cell wall hydrolytic enzymes, expansins, pathogenesis-related genes, and abscisic acid (ABA) and stress responsive genes. Further studies indicated that exogenous ABA was capable of promoting flower abscission in wild rice species and the SM transgenic plants. ABA from the developing embryo may have served as the signal for the initiation of rice grain abscission. The study suggests that ABA is the hormone that signals rice grain abscission and SM is a regulator in the signaling pathway. ACKNOWLEDGEMENT First and foremost, I would like to thank my advisor, Dr. Tao Sang, for his guidance and support through my academic career at Michigan State University. Dr. Sang has been an excellent and enthusiastic mentor, and without his support this research could not have been accomplished. I would like to express my great appreciation to my committee members, Dr. Robert Last and Dr. Shinhan Shiu for their guidance and encouragement. I benefited a lot from taking courses they taught. Dr. Last and Dr. Shiu have been always willing to help me whenever I need. I am grateful to the members of the Sang lab. Dr. Changbao Li offered me great help in RNA labeling and hybridization. Undergraduate assistant Angela Fowlkes-Sedillo spent a lot of time evaluating flower shattering in response of ABA treatment; Trevor Briggeman helped me a lot on greenhouse work. Graduate student Mike Grillo gave me considerable suggestions on my TA work. I would also like to thank Dr. Sheng Yang He and Dr. Beronda Montgomery-Kaguri for allowing me to use their equipment. I thank Dr. Jeff Landgraf for technical assistance on microarray experiment, and Anirban Mondal and Kaiyu He for studying my data for a class group project. I owe many thanks to my husband and daughter for their love and support. It is their understanding and encouragement that inspired me to accomplish this work. iii TABLE OF CONTENTS LIST OF TABLES ................................................................................ vi LIST OF FIGURES ................................................................................ vii INTRODUCTION ................................................................................... 1 Abscission Zone .................................................................................. 1 Positive Hormonal Regulation of Abscission .............................................. 2 Genes Up-regulated in the Abscission Process ........................................... 7 Genes Involved in the Grain Abscission During Rice Domestication .............. 10 MATERIALS AND METHODS ................................................................. 13 Rice Gene Transformation ................................................................... 13 DNA Isolation and Screening of Transgenic Plants ..................................... 15 Segregation Analysis .......................................................................... 16 Phenotypic Evaluation ......................................................................... 16 Oligo Microarray ................................................................................ 16 RNA Isolation and Quality Check ........................................................... 17 RNA Labeling .................................................................................... 18 Hybridization and Washing ................................................................... 18 Data Acquisition and Analysis ............................................................... 19 ABA Treatment .................................................................................. 20 Seed ABA Extraction and Analysis ........................................................ 20 RESULTS ........................................................................................... 22 RNA quality check .............................................................................. 22 Developing Transgenic Lines ................................................................ 23 Characterization of T0 Transgenic Plants ............................................ 23 Segregation Analysis of T1 Transgenic Plants ............................... ' ....... 2 4 Phenotypic Evaluation of T1 Transgenic Plants .................................... 25 Phenotypic Evaluation of 812-6 T2 Homozygous Transgenic Plants .......... 30 Microarray Analyses ............................................................................ 31 Comparison of Different Background Correction Methods ....................... 31 Genes Up-regulated by SM ............................................................. 33 Genes Down-regulated by SM ........................................................ 43 ABA Regulation of Abscission ............................................................... 45 Flower Shattering in Response of ABA Treatment .................................. 45 Evaluation of Seed ABA Content at Different Developmental Stages...47 DISCUSSION ...................................................................................... 49 Microarray Analysis ............................................................................. 49 Gene ExpresSion Regulated by SM ........................................................ 51 ABA regulation of Abscission ................................................................. 54 APPENDIX ......................................................................................... 57 Author’s Publication List During the Master’s Program ............................... 57 REFERENCES .................................................................................... 58 LIST OF TABLES Table 1. Media used for rice transformation ................................................ 15 Table 2. X2 test of T1 transgenic plants ...................................................... 24 Table 3. Number of up—regulated genes in various functional categories identified in three developmental stage .......................................................... 34 Table 4. Genes up-regulated by SM .......................................................... 34 Table 5. Number of genes down-regulated by SM ........................................ 44 Table 6. Genes down-regulated by SM ...................................................... 44 vi LIST OF FIGURES Figure 1. RNA integrity assessment ......................................................... 23 Figure 2. Grain shattering phenotype in line S12 T1 plants ............................ 26 Figure 3. Grain shattering phenotype in line S14 T1 plants ............................ 27 Figure 4. Grain shattering phenotype in line S15 T1 plants ............................ 28 Figure 5. Grain shattering phenotype in line S11 T1 plants ............................ 29 Figure 6. Grain shattering phenotype of the T2 plants derived from S12 ............. 31 Figure 7. MA-plots for one slide of stage 1 after different methods of background correction without any normalization ............................ 32 Figure 8. O. nivara flower shattering in response of ABA treatment .................. 46 Figure 9. Flower shattering of T2 transgenic plants and the control segregated from the same TO parent S12 in response of ABA treatment ............. 47 Figure 10. Seed ABA content in O. nivara .................................................. 48 vii INTRODUCTION Abscission refers to a developmental process that leads to the detachment of a plant organ. Abscission is a widespread phenomenon and is very important for the plant life cycle. Most deciduous plants abscise their leaves before winter, while evergreen plants continuously shed older leaves. A plant may drop its unwanted organs such as petals, sepals or filaments when they are no longer functionally essential. Fruits and seeds may be shed for seed dispersal. A plant may also abscise immature fmits to conserve resources needed to bring the remaining fmits to maturity. In response to disease or pathogen attack, a plant may shed its infected organs to protect the rest of the plant. Abscission also impacts agricultural productivity. Easy shedding of fruits causes considerable yield loss of crops. One of the essential steps of plant domestication was the reduction in fruit or seed shattering to allow effective field harvest. Important examples include the reduction of grain shattering in cereals and pod shattering of legumes. Abscission Zone Abscission consists of two developmental stages. The first is to form an abscission zone (AZ) and the second involves the response to environmental and hormonal signals that trigger cell wall hydrolysis in the abscission zone (Roberts et. al., 2002). The formation of the A2 at the location of organ separation may be completed months before organ detachment actually occurs. (Sexton and Roberts 1982; Gonzalez-Carranza et al., 1997). In dicotyledonous plants such as Arabidopsis, tomato, and bean, an abscission zone usually encompasses several layers of cells. In comparison to adjacent cells, AZ cells are smaller, contain denser protoplasm and larger deposits of starch, and have smaller intercellular spaces. Cell expansion in AZ often occurs during the abscission process (reviewed by Sexton and Roberts, 1982). In monocotyledonous plants such as in rice and oat, the AZ is located between the flower (or grain) and pedicel, and mostly consists of one layer of small, thin-walled cells. It is thus also called an abscission layer (Sargent 1984; Jin 1986). In rice, the abscission layer forms 16-20 days before flowers open for pollination (Jin 1986). Positive Hormonal Regulation of Abscission In 1955, Osborne published the first evidence that a diffusible substance in senescent leaves in beans and several other species accelerated abscission when applied to bean leaflet explants (Osborne 1955). Subsequently, several studies reported physiologically similar substances from leaves and fruits. Cams and his colleagues found a diffusible substance collected from the base of the cotton fruit (Gossypium hirsutum) promoted abscission (Carns 1958). This work led to the purification and partial identification of abscisin I, a weak abscission-accelerating crystallized substance isolated from mature fruit walls of cotton (Liu and Carns, 1961) Two years later, Ohkuma et al. identified and purified a strong abscission-accelerating acid from young cotton fruits and named it abscisin II (Ohkuma et al., 1963). The structure of abscisin H was then determined by Ohkuma et al. (1965). Shortly after, an abscission accelerator from the fruits of yellow lupin (Iupinus Iuteus) was identified and found to be identical to abscisin 11 (Porter and van Steveninck, 1966). However, the name abscisin II was called into question by some chemists because they concerned that a name such as abscisin 11 could raise difficulties for further naming analogues and derivatives. They favored a name that ends in “-ic acid”. At the Sixth international Conference of Plant Growth Substances held in Ottawa in 1967, a group of 12 scientists agreed to change the name to abscisic acid (ABA). This concluded the discovery and naming of the plant hormone, ABA, whose function, as indicated by its name, was thought to positively regulate plant organ abscission. ABA-accelerated abscission was observed in buds, leaves, pedals, flowers, and fruits (Addicott and Carns, 1983). ABA can also increase the rate of leaf senescence (Jackson and Osborne, 1972), promote ethylene synthesis (Riov et al., 1990), and induce cell wall hydrolytic enzymes such as cellulase (Craker and Abeles, 1969). However, shortly after its discovery, studies began to show that ABA is not particularly effective in promoting abscission. For example, the leaf abscission responses to ABA in Citrus were found to be season-dependent. Leaves sprayed with ABA in summer fell off, but those sprayed in winter did not (Cooper et al., 1968). Work by Jackson and Osborne suggests that ABA probably stimulates the abscission process through its ability to promote tissue senescence and ethylene climacteric (Jackson and Osborne, 1972). This is further supported by the result that ABA is not capable of speeding up abscission in Citrus if the tissue’s ability to produce ethylene is inhibited by aminoethoxyvinylglycine (AVG) (Sagee et al., 1980), suggesting that ABA functions through ethylene to accelerate abscission. These observations raised the question of whether ABA is the major abscission accelerator. The ABA’s role in regulating abscission was further obscured after the discovery of its essential roles in regulating dormancy and stomatal closure. When the role of ethylene in accelerating abscission was increasingly demonstrated in dicots, the role of ABA in regulating abscission was considered to be minor (Patterson 2001 ). Numerous studies have shown that ethylene can promote abscission of leaves, floral organs and fruits. Evidence mainly falls into four categories. First of all, there is a correlation between elevated ethylene production and the onset of abscission. Many fruits produce considerable amounts of ethylene in correlation with ripening and abscission (Pratt 1974; Walsh 1977). Natural ethylene or ethylene precursor level is often higher during fruit abscission (Brady et al., 1991; Burdon and Sexton, 1993). Further study of B-glucuronidase (GUS) expression driven by the 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (the enzyme that synthesizes the ethylene precursor ACC) promoter exhibits localized expression within the abscission zone (Ecker and Theologis, 1994). Second, abscission can be delayed by ethylene inhibitors. When ethylene production was inhibited by the antisense of ACC oxidase, the enzyme that converts ACC to ethylene, the activation of fruit abscission in melon was not activated and the fruit did not drop (Ayub et al., 1996). Likewise, the ethylene biosynthesis inhibitor, aminoethoxyvinylglycine (AVG), could reduce apple fruit drop (Kondo and Hayata, 1995). Third, abscission is delayed in ethylene insensitive mutants. Arabidopsis ethylene insensitive mutants etr1-1 (Bleecker et al., 1988) and ein2 (Guzman and Ecker, 1990) show significantly delayed shedding of floral parts (Bleecker and Patterson, 1997). Finally, ethylene can up-regulate the expression of cell wall degrading enzymes in the abscission zone (Boller et al., 1983; Taylor et al., 1990). Genes encoding these enzymes contain promoter elements that are subject to regulation by ethylene (Bleecker and Patterson, 1997). In the applied side, ethylene has been used as a chemical abscission agent for fruit harvest (reviewed by Addicott, 1982). Fruits can be shaken off from trees using mechanical devices when the abscission processes are well advanced by ethylene. This is particularly helpful for cultivars whose fruits are not yet dropping at the optimum harvest time and cannot be removed by the limited degree of shaking that a tree can tolerate. The best examples include the use of ethylene to help harvest of walnuts and pecans. Ethephon, a chemical that is easily converted to ethylene when absorbed by plants, has been effectively used for help harvest apples, cherries, plums, prunes, olives, oranges, and coffee berries. While ethylene has become the leading plant hormone in the basic research and agricultural applications related to abscission, researchers recently began to question how widespread the role ethylene plays as the primary hormone regulator of abscission. van Doom and Stead surveyed more than 300 flowering plant species and found that although in most dicots flower abscission was regulated by ethylene, most monocots showed ethylene-insensitive petal abscission (van Doom and Stead, 1997; van Doorn 2002). Work by Sexton et al. indicates that tulip tepal abscission does not respond to ethylene or ethylene antagonists (Sexton et al., 2000). Aneja et al. reported that during cocoa flower abscission, ABA levels increased dramatically prior to_abscission, while ethylene production only increased slightly. Furthermore, the ABA synthesis inhibitor, fluridone, inhibited the formation of an abscission zone and consequently the abscission or senescence of flowers; whereas the ethylene biosynthesis inhibitor, AVG, only slightly delayed but did not prevent abscission (Aneja et al., 1999). In addition, studies in grain abscission of wild oat (Sargent et. al., 1984) indicated that neither ethylene nor ACC promoted the cell separation process, and AVG did not delay abscission. In contrast, it was ABA that accelerated abscission of grains of wild oat. These observations raised the questions of whether the abscission of monocotyledons and dicotyledons is controlled by different hormones (Sargent et al., 1981, 1984), whether the abscission of different organs, such as leaf and fruit or flower is regulated by the same hormone, and whether the key regulator has yet to be determined (Roberts et. al., 2002). Genes Up-regulated in the Abscission Process Abscission occurs in multiple organs, such as leaf, flower, seed and fruit. The model plant Arabidopsis does not have leaf or fruit abscission, but it does display floral organ abscission (Patterson 2001). Progress has been made recently in studying Arabidopsis floral organ abscission, which led the identification of several genes with novel functions (reviewed by Lewis et al., 2006). Genes involved in abscission of other organs are relatively poorly understood. In general, at least two categories of genes are up-regulated in almost all organ abscission systems studied to date. The first group includes cell wall hydrolytic enzymes and expansins. During abscission process, cell wall degrading enzymes play vital roles to degrade middle lamella or even primary cell wall. The first enzyme observed to be involve in cell wall degradation at the site of abscission was B-1,4-glucanase, or cellulase (Horton and Osborne, 1967; Lewis and Varner, 1970; Sexton and Roberts, 1982). This gene belongs to a large gene family, and seven different isozymes (Cel1 to Cel7) have been cloned in tomato (reviewed by Roberts et al. 2002). During tomato flower abscission, an increased expression of Cel1, Cel2, and Cel5 has been detected (del Campillo and Bennett, 1996; Gonzalez-Bosch et al., 1997; Kalaitzis et al. 1999). But which, if any, of these isoforms may contribute to abscission is still unclear. Polygalacturonase (PG) is another abscission-related enzyme. The increased enzyme activity was reported during the abscission of leaves (Taylor et al., 1993), flowers (Tucker et al., 1984) and fruits (Bonghi et al., 1992; Henderson et al., 2001). It has been proved that in tomato three PG isoforms (TAPG1, TAPG2 and TAPG4) are associated with abscission. The GUS expression of TAPG1 and TAPGZ was localized specifically in the abscission zone of leaf petioles, flower and fruit pedicels, petal corolla, and stigma, and the expression was increased by ethylene and inhibited by IAA (Hong et al., 2000). In Arabidopsis, an abscission-related PG (PGAZAT) was shown to be specifically expressed at the base of the anther filaments (Gonzalez—Carranza et al., 2002). A null mutant of PGAZAT was found slightly delayed in ethylene-promoted flower abscission (Roberts et al., 2002). Expansins are associated with cell wall loosening. Two expansin genes have been cloned from the abscission zone region of ethylene-treated leaflets, and have been found to be specifically up-regulated during cell separation (Belfield et al. 2005). The second group included pathogenesis-related genes. Abscission provides an ideal site for pathogen invasion, and can be recognized as a classical defense response in many plants (Eyal et al., 1993). Pathogenesis-related (PR) genes are shown to express at a higher level during abscission. del Campillo and Lewis (1992a) found that abscission of the primary leaves of been was accompanied by the accumulation of a number of PR proteins. Such proteins were also accumulated in bean anthers and pistils during flower abscission .(del Campillo and Lewis, 1992b). Coupe et al. (1997) isolated several types of PR cDNA clones from ethylene treated leaflet abscission zone in Sambucus nigra. When Bleecker and Patterson fused the chitinase promoter to GUS and transformed the construct into Arabidopsis, the expression was observed at floral abscission zone (Bleecker and Patterson, 1997; Patterson and Bleecker, 2004). Plant defense associated enzymes, B-1,3-glucanases (Volko et al., 1998) and jasmonic acid biosynthetic enzymes (Kubigsteltig et al., 1999) were up-regulated specifically in the floral abscission zone region in response to different stresses. Metallothionein-like proteins also accumulate during ethylene-promoted abscission. These proteins might act to scavenge the free radicals within the abscission zone tissues (Coupe et al., 1995). Genes Involved in Grain Abscission During Rice Domestication Rice (Oryza Sativa), the world’s staple food, feeds more than half of the world’s population. Wild rice species shed their mature grains rapidly to ensure efficient seed dispersal. However, to allow effective field harvest, human must select those plants that can hold on to their ripe grains. Reduction in grain shattering is one of the first and most important agricultural traits selected by humans (Harlan 1975). The genetic basis of rice grain shattering remained unknown until recently. Two grain shattering genes, sh4 (Li et. al., 2006b) and qSH1 (Konishi et. al., 2006) were identified in rice through quantitative trait loci (QTL) mapping. qSH1 was cloned from a shattering- type cultivar and shown to encode a BEL1-type homeobox gene. A single-nucleotide polymorphism (SNP) in the 5’ regulatory region was found responsible for the loss of grain shattering. A near isogenic line (NIL) that contained the qSH1 region exhibited an improved abscission layer, suggesting qSH1 is involved in the development of abscission layer. SM is a grain shattering QTL identified from crosses between a wild rice species Oryza nivara and a cultivar of O. sativa ssp. indica (Li et al., 2006a). It was positionally cloned and the mutation responsible for the derivation of nonshattering in cultivated rice was mapped to a 1.7 kb region of a gene with a previously unknown function. Sequence analyses of additional species of wild rice and cultivars, along with gene transformation experiments confirmed that a single amino acid substitution from lysine to asparagine in the predicted MYB3-like DNA-binding domain caused the reduction of shattering in cultivated rice. The nuclear localization of sh4 protein, together with bioinformatic analysis, suggests the gene is a transcription factor. SM is required for the development of the abscission layer between a grain and pedicel that controls programmed cell separation. The amino acid substitution weakened the function of SM and caused the incomplete development of the abscission layer in cultivated rice. However, the mutation did not eliminate abscission layer function and allowed manual separation of the grains from the pedicel, a part of the rice harvest process known as threshing. The increased expression of sh4 in the late stage of seed maturation suggests that the gene may also be involved in the activation of the abscission process (Li et al., 2006b), which occurs after the formation of abscission layer. To test this hypothesis, one of the appropriate approaches would be to examine what genes are regulated by SM. Microarray analysis has proved to be a powerful tool to identify gene transcription profiles at the genome-wide level (Brown and Botstein, 1999; Young 2000; Lockhart and Winzeler, 2000; Harmer et. al, 2000). This sensitive and productive method should generate more information about changes in transcription during abscission process, and opens a door to further explore functions of abscission related genes. To identify genes regulated by SM during ll the grain abscission process, microarray analysis was conducted using sh4 transgenic plants and the controls. MATERIALS AND METHODS Rice Gene Transformation Mature rice seeds of a joponica cultivar, T309, were dehusked, surface-sterilized with 70% ethanol for 1 min, rinsed with sterile water 3 times and sterilized again with 20% Chlorox bleach for 20 min. After being washed 3 times again with sterile water, ten seeds were placed on each callus induction medium plate (Table 1) with sterilized forcep. Plates were sealed with parafilm and set under light at 30 °C for 3 to 4 weeks. On the day of the transformation, the proliferated calli derived from the scutella were divided into 2 mm pieces and transferred onto co-cultivation medium plates and each plate had about 100 pieces. One day before the transformation, Agrobacten'um tumefacias strain EHA 101 carrying sh4 construct 1, which harbored O. sativa promoter and lysine region of O. nivara allele (Li et al., 2006b) was grown in AB liquid medium (Chilton et al., 1974) containing 50 mg/L streptomycin overnight at 28°C with vigorous shaking at 250 rpm. Agrobacten'um cells were harvested by centrifugation at 3000 g for 10 min at 4°C, resuspended in PIM2 medium (Kant et al., 2001) supplemented with 100 pM acetosyringone and 50 mg/L hygromycin (Table 1). The Agrobacten'um cell cultures were diluted with PIM2 medium to ODsoo < 0.1, measured with a spectrophotometer (Barnstead ll Thermolyne Corporation, Dubuque, IA, USA). 13 Ten microliter of the diluted aliquot were pipetted onto a callus piece on co-cultivation medium (Table 1) and set in the dark at room temperature for 2-3 days. The cocultivated calli were washed with sterile water containing 250 mg/L cefotaxime (Sigma, St. Louis, MO, USA) to kill the Agrobacterium. The seeds were blotted dry on sterilized filter paper and transferred to selection medium (Table 1) containing 50 mg/L hygromycin. Plates were sealed with parafilm and put under continuous light at 30°C for 4 weeks. Fast growing calli on selection medium were transferred to 20x150 mm shoot induction medium plates (Table1), and set under 16-h-light/8-h-dark cycles at 30°C. Regenerated shoots were transferred to root induction medium (Toki 1997, Table 1) for 2-3 weeks. Regenerated plantlets were then transferred to sterilized soil in small pots with a plastic cover to maintain humidity. After one week the seedlings were transplanted to big pots and grown to maturity in a greenhouse with 16-h-light/8-h-dark cycles. Table 1. Media used for rice transformation Medium Composition YEP medium 10 g/L yeast extract, 109/L peptone, 5 g/L NaCl Sg/L, 50 mg/L streptomycin, 15 g/L agar, PH 7.5 AB medium 1xAB buffer (3 g/L KzHPO4, 1 g/L NaHzPO4), 1xAB salts (1 g/L NH4CI, 0.15 g/L KCI, 0.01 g/L CBCI2' 2H20, 2.5 mg/L FeSO4'7H20), 5 g/L glucose, 50 mg/L streptomycin, PH 7.2 Calli MS salts and vitamins 1, 30 g/L sucrose, 2 mg/L 2,4-D, 0.3 g/L induction casamino acids, 2.8 g/L proline, 6 g/L agarose, PH 5.8 medium Co-cultivation MS salts and vitamins‘, 30 g/L sucrose, 10 g/L glucose, 2 mg/L medium 2,4-D, 1 g/L casamino acids, 100 pM acetosyringone, 6 g/L agarose, PH 5.2 PIM2 1% glucose, 75 mM MES (PH 5.6), 2 mM NaPO4 buffer (PH 5.6), medium 1xAB salts (1 g/L NH4Cl, 0.15 g/L KCI, 0.01 g/L CaCl2' 2H20, 2.5 mg/L FeSO4'7H20) Selection MS salts and vitamins‘, 30 g/L sucrose, 0.3 g/l casamino acids, 2.8 medium g/L proline, 2 mg/L 24-0, 50 mg/L hygromycin, 250 mg/L cefotaxime, 6 g/L agarose, PH 5.8 Shoot MS salts and vitamins‘, 30 g/L sucrose, 30 g/L sorbitol, 2 g/L induction casamino acids, 2 mg/L benzylaminopurine, 0.05 mg/L medium naphthylacetic acid, 50 mg/L hygromycin, 4 g/L gelrite, PH 5.8 Root MS salts and vitamins‘, 30 g/L sucrose, 50 mg/L hygromycin, 2 g/L induction gerite, PH 5.8 medium 1(Murashige and Skoog, 1962, purchased from SIGMA-ALDRICH, Inc., St. Louis, MO, USA.) DNA Isolation and Screening of Transgenic Plants DNA was isolated from leaf tissues of 10-day old seedlings as described previously (Li et al., 2006b). Transgenic plants were first screened by plasmid primers, Lac F (5’-TGGAGCTCCAGCI l l lGTTC-3’) and Lac R (5’-AGTTAGCTCACTCATTAGGC- 3’). Plants that failed to amplify the Lac band were further screened by coding region primers, ssh4-7819sF (5’-GAGAGCGCGTCGTAGACCTC-3’) and ssh4-8201sR (5’-GCAAGGGGACTGGACGCTG-3’) to rule out the possibility of the Lao negative band being caused by low DNA quality. Segregation Analysis Ten-day-old T0 and T1 seedlings were grown in small pots in a tray, and plants containing the transgene were identified by PCR with the above primers. Segregation ratios were calculated by counting the number of plants carrying the SM transgene and those lacking the transgene. Phenotypic Evaluation Phenotype was evaluated by measuring the force required to pull away flowers or grains from the pedicel (Li et al., 2006b). Five panicles of each plant were evaluated. Oligo Microarray Expression profiling was conducted with the 45K NSF rice oligo nucleotide array (http://www.ricearray.org/index.shtml), which consisted of two slides and was printed with 43,482 oligos designed for 45,116 TIGR V3 rice gene models from the TIGR Rice Annotation Database that have EST and/or full-length cDNA support. These oligos were cross referenced to the Kikuchi full-length cDNA dataset (Kikuchi et al., 2003) and were designed using PlCKY—2 (Chou et al., 2004). In addition to the rice gene oligos, 456 hygromycin resistant gene oligos were randomly spotted as controls. These hygromycin oligos served as positive controls in our case. RNA Isolation and Quality Check Total RNA was isolated and purified from tissues at the junction of flower/grain and pedicel using the Qiagen Plant RNeasy Kit (Qiagen Sciences, Germantown, MD, USA). Four developmental stages, pollination (flowering stage), grain soft dough (grain is less than half full), grain hard dough (grain is full but still green), fully mature grain (grain is full and yellow) were assayed. Tissues from three to five plants were pooled for each sample. RNA was prepared from three biological replicates for each developmental stage and was examined by an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) for integrity and NanoDrop spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA) for purity. To ensure data quality, only samples with well-defined 188 and 288 rRNA bands, and with A260/A280 ratios of 1.9-2.1 were included in the microarray analyses. RNA Labeling Because of the limited amount of tissues, fluorescent cRNA was generated using an Agilent Low Input Fluorescent Linear Amplification Kit (Agilent Technologies, Inc., Wilmington, DE, USA) with modifications. Briefly, both first and second strand cDNA were synthesized by incubating 450 ng total RNA with 1.2 pl of T7 promoter primer in nuclease-free water at 65 °C for 10 min followed by incubation with 4 pl of 5x first strand buffer, 2 pl of 0.1MDTT, 1 pl of 10mM dNTP, 1 pl of 200 U/pl MMLV RT, and 0.5 pl of 40Ulul RNaseOUT at 40 0C for 2 h. Incubate samples at 65 °C for 15 min to inactivate MMLV RT. Immediately following cDNA synthesis, the reaction mixture was incubated with 2.4 pl of 10 mM cyanine-3- or cyanine-5-CTP (Agilent), 15.3 pl of nuclease-free water, 20 pl of transcription buffer, 6 pl of 0.1M DTT, 8 pl of NTP mix, 6.4 pl of 50% PEG, 0.5 pl of RNaseOUT, 0.6 pl of inorganic pyrophosphatase, and 0.8 pl of T7 RNA polymerase at 40 °C for 4 h. Cyanine 3 (Cy3) or cyanine 5 (Cy5) labeled cRNA was purified with Qiagen’s RNeasy mini spin columns. Amplified cRNA was quantified using a NanoDrop spectrophotometer. Only cRNA with a concentration higher than 1.5 pg/pl and the specificity higher than 8.0 pmol Cy3 or Cy5 per pg cRNA was used for hybridization. Hybridization and Washing Prior to hybridization, microarray slides were immersed in pre-hyb buffer (185 ml ddH20, 62.5 ml 20XSSC, 2.5 ml 10% SDS and 2.5 9 BSA) at 42 °C for 45 min, and washed in 0.1XSSC and ddH20. Mixed Cy3 and Cy5 labeled cRNA was dried and resuspended in 4 pl 10 mM EDTA. One microliter of 10 mg/ml yeast tRNA was added to the reaction mix as the block solution. The mixture was heated at 95 0C for 10 min, and 60 pl of pre-warmed SlideHyb buffer (Ambion, Austin, TX, USA) was added. The total of 64 pl mixture was applied to the array and hybridized at 48 0C for 16 h. After hybridization, the array was washed sequentially with solution I (1 XSSC and 0.2% SDS in ddH20), solution ll (0.1x SSC and 0.2% SDS in ddH20), and solution Ill (0.1XSSC in ddH20). Data Acquisition and Analysis Dried arrays were immediately scanned with an Affymetrix 428TM Array Scanner (Affymetrix, Inc., Santa Clara, CA, USA), and TIFF images were processed with GenePix pro 3.0 software. Spots with aberrant morphology were manually flagged. Raw data GPR files generated from GenePix were uploaded in R-based IimmaGUl package (Wettenhall and Smyth, 2004). Hybridization quality of each slide was checked with image array plot before normalization was performed. Various background correction methods were compared, and no background correction option was finally adopted. Scale normalization method was used. Toptable for differentially expressed genes was generated with FDR adjusted method. For the three replicates, a spot was removed from further analysis if any one replicate has flagged value, or any two of the three replicates has 40 percent feature pixels with intensities lower than two standard deviations above the background pixel intensity in both channels. With a cutoff of p value < 0.05 and two fold change, both up-regulated and down-regulated genes were identified for three developmental stages. ABA Treatment Flowering panicles from O. nivara and T309 transgenic plants were treated with 0.1 mM, 1.0 mM abscisic acid (A.G. Scientific, Inc., San Diego, CA, USA) and H20, respectively. Percentage of flower shattering was calculated every 24 hr by counting the number of flowers dropped when panicles were tapped by hand. Eight to ten panicles from at least three plants were examined for each treatment. Stigmas of a portion of unopened flowers on O. nivara panicles were removed. The panicles were observed for 30 days on plants and then removed and treated with 1.0 mM ABA. Seed ABA Extraction and Analysis Forty grains of O. nivara were sampled from four different developmental stages, Milky endosperm (grain less than half full), soft dough (grain half to 3/4 full), green hard dough (grain full but still green) and yellow hard dough (grain fully full and yellow). Grains were dehusked and ground to power in a mortar under liquid nitrogen, and 5 ml ABA extraction buffer (100 mg/L 2,6-ditert-butyl-methyl phenol, 20 500 mg/L citric acid in 80% methanol) added, rotated at 4°C in the dark for 16h. The suspension was centrifuged at 1000x g at 4°C for 20 min and then transferred to four new microcentrifuge tubes and vacuum dried. The dried residue was dissolved with 100 pl of Tris saline buffer (TBS, 25mM Tris, 100mM sodium chloride, 1mM magnesium chloride hexahydrate) containing 0.02% sodium azide, and then the extracted ABA from the four tubes was combined together. Quantitative analysis of ABA was performed by the indirect enzyme-linked immunosorbent assay (ELISA) method, using the Phytodetek-ABA-Kit as described by the manufacturer (Sigma-Aldrich, Inc., St. Louis, MO, USA), and measured with a Spectra Max M2 microplate reader (Molecular Devices Corporation, Sunnyvale, CA, USA). 21 RESULTS RNA Quality Check RNA samples were assessed quantitatively and qualitatively by the Nanodrop spectrophotometer. If an A260/A280 ratio was below 1.9, the extraction procedure was repeated. RNA samples with accepted A260/A280 ratio were further analyzed using an Agilent bioanalyzer (Figure 1). The 183 and 285 peaks were clearly visible at 41 and 47 seconds respectively in stage I, II and Ill, whereas 28s and 18s peaks were absent in stage IV. The result indicates RNA samples from the Stage I, II and III were qualified for microarray. The total RNA from stage IV was almost degraded for the reason of seed maturation. Thus stage IV could not be included in the microarray experiment. 22 16 Sta el . g 30‘. Sta ell , 8 12 . 9 285i: 3 , 9 28s,! , § I ' I "20‘ 18sl I I II ‘ ‘ I' I 8 8‘ . 185’ 3 I I ‘ i 8 * l I! I :10, ‘ I 3 i; . > ' I ‘ I LT. 44 I I fit”) IN“ I I * I I. ' I I, I 4 -. h I‘, \ 1 JL/ ‘— ‘ '1 “""“" "' “"\.__ I “It 1 “MW“ \k I ....._.._., ,...,.......~.--....,.-._-..,.....,.. , I ‘ . . 1 Y r . v . . 20 3o 40 50 60 ' 20 3o 40 50 60 25 . 25 Z _ . stage III 285]! , I , Stage IV , a) I II I I ' I g A 18 III I I . q, 15' I g j , , 3 , j l j . g : I I I i 3 5 ,I — l LL M s I 20 30 4o 50 60 2'0 ' 3b 4b 50 ' 60 Second Second Figure 1. RNA integrity assessment. The purified total RNA samples from different developmental stages were measured by Agilent 2100 Bioanalyzer and the results were presented as Electropherograms. RNA of one biological replicate from each stage was presented. Developing Transgenic Lines Characterization of T0 Transgenic Plants sh4 construct 1 which harbored O. sativa promoter and most of the coding region of O. nivara that contains the functional SNP was transformed into an O. sativa ssp. Japonica cultivar, T309. DNA was extracted from transgenic plants, and PCR analysis indicated that all of the transgenic plants carried the SM transgene. 23 In comparison with the control, most of T309 transgenic lines (T0) had loosened flowers (Li et al., 2006b). Four transgenic lines with the clear phenotype, S11 , S12, S14, and S15, were grown for advanced generations, T1, T2, and so on. Segregation Analysis of T1 Transgenic Plants DNA was extracted from 10—day-old T1 seedlings derived from T0 plants through self-fertilization. PCR analysis was performed to determine the presence of the SM transgene. The transgene segregated in all four lines, and the Chi square ( )6) test indicated that the transgene was introduced into each of the four T0 transgenic lnes at a single locus (P>0.05) (Table 2). Table 2. )6 test of T1 transgenic plants Line Observed number Expected number X2 P value With without with without transgene transgene transgene transgene S11 67 25 69 23 0.232 0.630 S12 65 23 66 22 0.061 0.805 S14 54 19 55 18 0.074 0.786 S15 68 15 62 21 2.295 0.130 Df=1 24 Phenotypic Evaluation of T1 Transgenic Plants The phenotype of all T1 transgenic plants grown under the same conditions in the greenhouse was evaluated by measuring the force required to pull away the mature grains from the pedicels (Li et al., 2006a). Compared with plants without the trangene, the force required to break away the grains of plants carrying the transgene was greatly reduced (Figure 2-5). All four lines showed significant difference of grain shattering between transgenic plants with and without the trangene (p = 0.004, 0.002, 0.001 and 0.023 for S11, S12, S14 and S15, respectively; student’s t-test). The variance of the measurement was relatively large due to the limited number of grains available from individual plants for the phenotypic evaluation. 25 200 . l 160 . T _, - -- T a -- 7512M 9 T r o _ T Ll. 80. * 4o. . .5; * luff}! .. ‘— N ('0 ‘— N 0") W' In (D N (D O) O 2 Z 2 o': 0'1 cx'n N o': 0': o'n cx'n 0'1 ‘7 U) (D (D (D (D CD CO U) (D U) (D (D a") Figure 2. Grain shattering phenotype in line 812 T1 plants. Grey bars indicate plants carrying sh4 transgene, black bars indicate plants that segregated from the same T0 parent and did not carry the transgene. The average force with standard deviation required to pull away a grain from the pedicel is illustrated. Plants marked with * were selected to grow for further segregation and phenotypic evaluation. Total number of grains evaluated was between 30 and 100, depending on the number of grains available at the time of measurement. 26 160- _ 1 1 -—-— 120 T :93 g * 080 u.- a: 1 40- gggw¢¢¢9©fi°9°92ssr gggiiiiiiiiiéfié wwwwwwwwwwwwawa Figure 3. Grain shattering phenotype in line S14 T1 plants. Grey bars indicate plants carrying sh4 transgene, black bars indicate plants that segregated from the same T0 parent and did not carry the transgene. The average force with standard deviation required to pull away a grain from the pedicel is illustrated. Total number of grains evaluated from each plant varied between 40 and 100, depending on the number of grains available at the time of measurement. Plants marked with * were grown for further phenotypic evaluation. 27 200 - Force (9) 80- 4o- S15N1 S15N2 S15N3 S15-1 S15-2 S15-3 S15—4 S15-5 fiv Figure 4. Grain shattering phenotype in line 815 T1 plants. Grey bars indicate plants carrying sh4 transgene, black bars indicate plants that segregated from the same T0 parent and did not carry the transgene. The average force with standard deviation required to pull away a grain from the pedicel is illustrated. Total number of grains evaluated was between 20 and 50, depending on the number of grains available at the time of measurement. 28 200 ‘ 160 Force (9) BOT 40- * S11-4 S11N1 S11N2 S11N3 S11-1 S11-2 S11-3 Figure 5. Grain shattering phenotype in line 811 T1 plants. Grey bars indicate plants carrying sh4 transgene, black bars indicate plants segregated from the same T0 parent and did not carry the transgene. The average force with standard deviation required to pull away a grain from the pedicel is illustrated. Plants marked with * were grown for further phenotypic evaluation. Total number of grains evaluated was between 20 and 50, depending on the number of grains available at the time of measurement. Among the T1 transgenic lines, plant S12-6 showed the best phenotype. Thus transgenic and the control seeds randomly selected from S12-6 were grown for 29 further analysis (double * in Figure 2). Meanwhile, two other plants from line 812 and three easiest-shattering plants from line S11 and S14 were chosen for further phenotypic evaluation. DNA was extracted from 10-day-old seedlings of T2 plants derived from the T1 line S12-6. PCR analysis from 32 T2 individuals demonstrated that the transgene was no longer segregating, indicating that this line was homozygous at the transgene locus. Phenotypic Evaluation of 812-6 T2 Homozygous Transgenic Plants The T2 plants derived from S12-6 showed an easy shattering phenotype (Figure 6). The plants segregated from the S12 T0 line that did not carry the transgene showed non-shattering phenotype. The Student’s t-test indicated a highly significant difference between the phenotypes (p<0.0001). Therefore, the T2 plants derived from homozygous transgene line 812-6 and the line without trangene were used for the microarray analysis. 30 q 200_ 160. 120- § 880- L— o -: LL 40‘ Ill llllllll IllllFr-v-r-r- sassassasttsasseseseeee ggggggggggfififififififigchécfichm wwwwwmwmmwwwwwmmwwfiwfifia Figure 6. Grain shattering phenotype of the T2 plants derived from 812. Grey bars indicate plants carrying sh4 transgene (from T1 line S12-6), black bars indicate control plants (from T1 line S12N2) that did not carry the transgene. The average force with standard deviation required to pull away a grain from the pedicel is illustrated. Fifty grains from each plant were evaluated. Microarray Analyses Comparison of Different Background Correction Methods For the raw data generated from genepix, several background correction methods, including None, Subtract, Half, Minimum and RMA were compared in 31 R-based Limma package (Smyth 2004) (Figure 7). a-t1 1 oy5(40)c 1 1 ey3(45) 11-11 1 Cy5(40)c1 1 Cy3(45) 6 8 1O 12 14 16 Background correction “Subtract" No background correction method a-t1 1 cy5(40)c1 1 Cy3(45) a-t1 1 Cy5(40)c1 1 Cy3(45) Background correction “Minimum" Background correction “RMA” method method Figure 7. MA-plots for one slide of stage 1 after different methods of background correction without any normalization. MA-plots for other slides were similar for each of these background correction methods. From the log-ratio M and log-intensity A plot (MA-plot), we can see that the “Subtract” method was not suitable for our dataset, which may be due to many negative values after background subtraction. These values cannot be used for 32 further analysis since Log cannot deal with negative values, so they were treated as missing observations. The “Minimum” and the “Half” (graph not shown) methods did not give better results as they assign some fixed positive values for the nonnegative intensities after subtracting the background intensities. The RMA method gave some normal plots. However, this method still showed biased distribution for our dataset. Based on the comparison of the MA-plot, no background correction seems to generate the least bias, thus it was adopted in our microarray data analysis. Genes Up-regulated by SM With a cutoff of two fold change and a p value of 0.05, a total of 278 genes were identified as differentially expressed in sh4 transgenic and control plants for three developmental stages. Two hundred and forty one genes were up-regulated by SM (Table 3). They were grouped into the following functional categories (Table 4). 33 Table 3. Number of up-regulated genes in various functional categories identified in three developmental stages Functional category Stage 1 Stage 2 Stage 3 Cell wall hydrolytic enzymes and expansins 9 0 1 Pathogenesis— related genes 15 2 2 ABA/stress related genes 10 3 6 DNA binding/transcription factor 3 0 3 Signal transduction 3 0 2 Membrane protein 4 0 2 Transport 3 0 4 Protein biosynthesis/modification 16 0 5 Metabolism 11 3 9 Photosynthesis 23 3 4 Others 29 4 22 Unknown 38 7 1 2 Table 4. Genes up-regulated by SM Category Annotation lD Stage Cell wall hydrolytic enzymes and expansins Alpha-expansin 11 precursor, putative LOC_OsO6950400 1 Expansin-related protein 2 precursor, putative LOC_0309929710 1 Mannan endo-1,4-beta-mannosidase, putative LOC_OsO1g47400 1 Pectinesterase family protein LOC_OsO3g28090 1 Endo-beta-mannanase, putative LOC_OsO3g61270 3 Glycosyl hydrolases family 16 protein LOC_OsO4gS3950 1 Glycosyl hydrolase fami|y1 protein LOC_OsO3g11420 1 Hydrolase, alpha/beta fold family protein, putative LOC_OsOBg27110 1 Glycosyl hydrolase family 14 protein LOC_Os10932810 1 Beta-fructofuranosidase 1 precursor, putative LOC_OsOZgO1590 1 Pathogenesis-related Glycosyl hydrolases family 18 protein LOC_Os10928080 1 Endochitinase A precursor, putative LOC_OsO4941620 1 26 kDa endochitinase 1 precursor, putative LOC_OsOSg33150 1 Chitinase 1 precursor, putative LOC_Os10928120 1 34 Table 4 cont’d Endochitinase A precursor, putative Pectinesterase inhibitor domain containing protein Pectinesterase inhibitor domain containing protein Polygalacturonase inhibitor 1 precursor, putative Win1 precursor, putative Xylanase inhibitor protein 1 precursor, putative Xylanase inhibitor protein 2 precursor, putative Glucan endo-1,3-beta-glucosidase 5 precursor Glucan endo—1,3-beta-glucosidase GV, putative Lectin precursor, putative Metallothionein-like protein type 3, putative P21 protein, putative Pathogenesis-related protein Bet v I family protein Pathogenesis-related protein Bet v | family protein ABA/Stress response Hsp20/alpha crystallin family protein Salt stress-induced protein Wound/stress protein Wound induced protein Wound induced protein, putative Universal stress protein family protein Late embryogenesis abundant protein ABANVDS induced protein Late embryogenesis abundant protein Lea14-A, putative ABA/WDS induced protein Stress responsive protein, putative Universal stress protein family protein BURP domain containing protein 16.9 kDa class I heat shock protein, putative Zinc finger A20 and AN1 domains containing protein At2936320 Dehydrin family protein DNA binding/transcription factor Floral homeotic protein APETALA1, putative Homeobox domain containing protein LlM domain containing protein 35 LOC_OsO4g41 680 LOC_OsO3g61 530 LOC_OsOSg46530 LOC_OsO7g38130 LOC_Os11g37970 LOC_OsO7g43820 LOC_OsOSg1 5770 LOC_OsO6g39060 LOC_OsO1g71810 LOC_OsO4909390 LOC_OsO5g11320 LOC_OsO3g4607O LOC_OsO4g39150 LOC_OsO4g391 50 LOC_OsO1gO4350 LOC_OsO1gZ4710 LOC_OsOZg51 710 LOC_OsO4954240 LOC_OsO4954300 LOC_0305928740 LOC_0305929930 LOC_Os1 1906720 LOC_OsO1g12580 LOC_OsO4934600 LOC_OsO1gO1450 LOC_0305937970 LOC_OsO5g1 2640 LOC_OsO1gO4380 LOC_OsO7gO7350 LOC_Os11926790 LOC_OsO3gS4160 LOC_OsO494581 0 LOC_Os10935930 ...\ “NANQAAAAAA—x—x‘ _x_x A—t—lNN—L—‘LA —L CONCOOOOOCO (JO Table 4 cont’d AP2 domain containing protein LOC_Os09935010 MYBG, putative LOC_0305935500 DNA-binding protein S1 FA2, putative LOC_OsO4g33440 Signal transduction EF hand family protein LOC_OsO6947640 EF hand family protein LOC_0305931620 EF hand family protein LOC_OsO2g39380 Res-related protein Rab-2-B, putative LOC_0302937420 Protein kinase domain containing protein LOC_Os12941090 Membrane protein Outer membrane lipoprotein blc precursor, putative LOC_OsO2g39930 Plant integral membrane protein TIGR01569 containing protein LOC_OsO4921320 16kDa membrane protein, putative LOC_OsO4933830 Plasma membrane ATPase 1, putative LOC_OsO7909340 Secretory carrier membrane protein family protein LOC_OsO5942330 Pyrophosphate-energized vacuolar membrane proton pump, putative LOC_OsO6gO8080 Transport Aquaporin RWC3, putative LOC_OsO4g47220 Aquaporin TIP-type RB7-18C, putative LOC_OsO6g22960 Aquaporin TlP3.1, putative LOC_0505914240 POT family protein LOC_OsO1965100 Nonspecific lipid-transfer protein precursor, putative LOC_OsO196074O Adaptin N terminal region family protein LOC_Os11907280 Major intrinsic protein LOC_OsO1913130 Protein biosynthesis/modification Eukaryotic aspartyl protease family protein LOC_OsO1941550 Eukaryotic aspartyl protease family protein LOC_OsO3908790 Eukaryotic aspartyl protease family protein LOC_OsO4958840 Ubiquitin-conjugating enzyme E2-17 kDa, putative LOC_OsO3957790 Subtilisin N-terminal Region family protein LOC_OsO7939020 Ubiquitin-conjugating enzyme E2-17 kDa, putative LOC_OsO4957220 Bowman-Birk serine protease inhibitor family protein LOC_OsO3960840 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase, putative LOC_Os12942884 60$ ribosomal protein L12, putative LOC_OsO2947140 36 0000 _x 00.3de 0000—8000044 w—t—L—s A“—t—t—A—t—L 00 Table 4 cont’d 6OS ribosomal protein L31, putative 4OS ribosomal protein SA, putative 60$ ribosomal protein L15, putative Ribosomal protein L36 containing protein 408 ribosomal protein S8, putative 408 ribosomal protein S10, putative 60$ ribosomal protein L6, putative 60$ ribosomal protein L4, putative 608 ribosomal protein L22-2, putative 4OS ribosomal protein S3a, putative Ribosome associated membrane protein RAMP4 containing protein 608 ribosomal protein L17, putative, expressed Metabolism Triosephosphate isomerase, cytosolic, putative Cytochrome P450 family protein Nucleoside diphosphate kinase 1, putative Fructose-bisphosphate aldolase, cytoplasmic isozyme ATP synthase subunit C family protein Enolase, C-terminal TIM barrel domain containing protein Geranylgeranyl hydrogenase, putative Glutathione S-transferase GSTU6, putative Oxidoreductase, 2OG-Fe oxygenase family protein Annexin-like protein RJ4, putative GDSL-like Lipase/Acylhydrolase family protein Farnesyl pyrophosphate synthetase, putative NAD dependent epimerase/dehydratase family protein NAD dependent epimerase/dehydratase family protein Heme oxygenase 1, putative 2-Hydroxyisoflavanone dehydratase, putative Gibberellin 2-beta-dioxygenase, putative Tropinone reductase, putative Alcohol dehydrogenase class III, putative Cyanate hydratase, putative SAM dependent carboxyl methyltransferase family protein 37 LOC_0302948660 LOC_OsO3908440 LOC_OsO3940180 LOC_OsO3959720 LOC_OsO4928180 LOC_OsO4935090 LOC_OsO4939700 LOC_OsO7908330 LOC_OsO794771 0 LOC_0803910340 LOC_OsO7939400 LOC_0308941810 LOC_OsO1905490 LOC_OsO4940470 LOC_OsO7930970 LOC_0308902700 LOC__Os12934110 LOC_OsO391 5950 LOC_OsO2951080 LOC_Os10938740 LOC_OsO1970930 LOC_0302951 750 LOC_Os10932580 LOC_OsO1950760 LOC_OsO7940690 LOC_OsO6944180 LOC_OsO6940080 LOC_Os09928630 LOC_OsO1955240 LOC_Os1 1925700 LOC_0302957040 LOC_Os10933270 LOC_0802948770 w—LAAA—K—L—L—K—A 0000 (pa—3.11.; wwwwé—KQQNN-‘ww-‘A N Table 4 cont’d O-methyltransferase family protein Photosynthesis Chlorophyll a-b binding protein 2, chloroplast precursor, putative Chlorophyll a-b binding protein CP24 1OB, chloroplast precursor, putative Chlorophyll a-b binding protein of LHCII type III, chloroplast precursor, putative Chlorophyll a-b binding protein 6A, chloroplast precursor, putative Chlorophyll a-b binding protein 8, chloroplast precursor, putative Chlorophyll a-b binding protein 7, chloroplast precursor, putative Chlorophyll a-b binding protein 8, chloroplast precursor, putative Thioredoxin F-type 2, chloroplast precursor, putative Ferredoxin-NADP reductase, leaf isozyme, chloroplast precursor, putative Elongation factor Tu, chloroplast precursor, putative Chloroplast 3OS ribosomal protein S10, putative Acyl-desaturase, chloroplast precursor, putative Photosystem I reaction center subunit psaK, chloroplast precursor, putative Ferredoxin-1, chloroplast precursor, putative Photosystem l reaction center subunit V, chloroplast precursor, putative Photosystem l reaction center subunit VI, chloroplast precursor, putative Ribulose bisphosphate carboxylase/oxygenase activase, chloroplast precursor, putative Expressed protein;Photosystem l reaction center subunit Ill, chloroplast precursor, putative Oxygen-evolving enhancer protein 3-1, chloroplast precursor, putative Ferritin 1, chloroplast precursor, putative Possible Photosystem ll reaction center Psb27 protein, putative 38 LOC_Os10902880 LOC_OsO1941710 LOC_OsO493841 0 LOC_OsO7937550 LOC_OsO6921 590 LOC_OsO2910390 LOC_OsO7938960 LOC_OsO2910390 LOC_OsO1968480 LOC_0302901340 LOC_0302938210 LOC_OsO391 0060 LOC_OsO3930950 LOC_OsO7905480 LOC_0308901380 LOC_0309930340 LOC_OsO5948630 LOC_Os11947970 LOC_OsO3956660 LOC_OsO7936080 LOC_Os12901530 LOC_OsO3921560 1,2,3 1,3 u—L A—k—‘L—K 1,2,3 Table 4 cont’d Ribulose bisphosphate carboxylase large chain precursor, putative Ribulose bisphosphate carboxylase large chain, catalytic domain containing protein, putative SOS ribosomal protein L12-2, chloroplast precursor, putative Others Histone H4, putative Histone H4, putative Histone H3, putative Histone H4, putative Histone H4, putative Histone H2A, putative Histone H4, putative Peroxidase 21 precursor, putative AhpC/T SA family protein Protein phosphatase 2C, putative Peptidyl-prolyl cis-trans isomerase 1, putative RNA recognition motif family protein Fasciclin domain containing protein Cytidine and deoxycytidylate deaminase zinc-binding region family protein ADP-ribosylation factor, putative GTP-binding nuclear protein Ran/T C4, putative RPT2, putative, expressed Cupin family protein, expressed Shrunken seed protein, putative Pollen-specific protein C13 precursor, putative B-type cyclin, putative Fasciclin-like arabinogalactan protein 1 precursor, putative Dof domain, zinc finger family protein CP12, putative ER6 protein, putative MTERF family protein Yippee, putative Enzyme of the cupin superfamily, putative 39 LOC_Os10921280 LOC_0306939730 LOC_OsO1947330 LOC_OsO4949420 LOC_0302945940 LOC_0306906460 LOC_OsOQg38020 LOC_0309926340 LOC_0505938640 LOC_OsO3902780 LOC_OsO7949360 LOC_OsO1948420 LOC_OsO5938290 LOC_Os06949480 LOC_OsO3961990 LOC_OsO1906580 LOC_OsO1951 540 LOC_OsO1959790 LOC_OsO5949890 LOC_Os11902610 LOC_030890341 0 LOC_OsO2903070 LOC_OsO4932680 LOC_0302933330 LOC_0302949420 LOC_0302949440 LOC_OsO391 9380 LOC_0302952314 LOC_OsO3924590 LOC_OsO39491 50 LOC_OsO4936760 A—LN—L—kwwA wwww—t—waw—tAAA 4.x _x_x|\)oo_s_s_x Table 4 cont’d LOC_0308902690 LOC_Os089091 80 LOC_Os11905190 LOC_0306904990 LOC_0308928790 LOC_0306934450 LOC_0309924540 LOC_0305924560 LOC_0805935740 LOC_Os05945450 LOC_Os11905930 LOC_OsO3906360 MA3 domain-containing protein, putative Postsynaptic protein Cript, putative Phytosulfokines 2 precursor, putative Early nodulin 93, putative Dirigent—like protein Zinc finger, C3HC4 type family protein PPIC-type PPIASE domain containing protein NC domain-containing protein, putative Pi starvation-induced protein, putative Nuclear protein, putative CCT motif family protein Seed maturation protein :al Meristem protein LOC_OsO4938720 ARI, RING finger protein, putative LOC_0808942740 DNA-directed RNA polymerase alpha chain, putative LOC_OsO1957944 Tubulin beta-2 chain, putative LOC_OsO3901530 Actin-related protein 2/3 complex 34kDa subunit family, putative LOC_OsO4943290 Retrotransposon protein, putative, Ty3-9ypsy subclass LOC_OsO1952690 Transposon protein, putative, CACTA, En/Spm sub-class LOC_OsO1969020 atrotransposon protein, putative, Ty3-9ypsy subclass LOC_Os03919600 Transposon protein, putative, CACTA, En/Spm sub-class LOC_OsO7923640 Transposon protein, putative, CACTA, En/Spm sub-class LOC_OsO4934170 Transposon protein, putative, CACTA, En/Spm sub-class LOC_OsO794891 0 Retrotransposon protein, putative, unclassified LOC_0508921960 Retrotransposon protein, putative, Ty3-9ypsy subclass LOC_OsO1913650 Unknown AAwwwwwwoooowooN-a—s-a A OD Expressed protein Expressed protein Expressed protein Expressed protein Expressed protein Expressed protein 40 LOC_OsO1906620 LOC_OsO1910400 LOC_OsO1932770 LOC_OsO1942520 LOC_0302903510 LOC_0302903520 .s_x_x_s_s[\) Table 4 cont’d Expressed protein Expressed protein Expressed protein Expressed protein Expressed protein Expressed protein Expressed protein Expressed protein Expressed protein Expressed protein Expressed protein Expressed protein Uncharacterized plant-specific domain TlGR01570 family protein Expressed protein Hypothetical protein Expressed protein Hypothetical protein Expressed protein Expressed protein Expressed protein Uncharacterized plant-specific domain TIGR01568 family protein Expressed protein Expressed protein Expressed protein Expressed protein Expressed protein Expressed protein Expressed protein Expressed protein Expressed protein Expressed protein Expressed protein Expressed protein Expressed protein Expressed protein 41 LOC_OsO2903710 LOC_OsO2945930 LOC_OsO3905520 LOC_OsO3914570 LOC_OsO3939830 LOC_OsO3942600 LOC_OsO3947270 LOC_0503948626 LOC_OsO3948626 LOC_0503958850 LOC_OsO4933310 LOC_Os04933710 LOC_OsO4954600 LOC_OsO7902340 LOC_OsO7905500 LOC_Os0790581O LOC_OsO7925320 LOC_OsO7931490 LOC_OsO7937240 LOC_OsO7943020 LOC_OsO79481 50 LOC_Os10934270 LOC_OsO6909900 LOC_050691 5400 LOC_OsO691 7450 LOC_030591 0800 LOC_OsO5930400 LOC_0305933280 LOC_Os12935470 LOC_Os11931400 LOC_OsO8939330 LOC_Os11902330 LOC_OsO3957220 LOC_Os109391 50 LOC_OsO4954230 .3 . A—k—t—A—k—LwAANAA .34 NNNAAANNA—LAAAAA Table 4 cont’d Expressed protein LOC_OsO1913660 Expressed protein LOC_OsO1949210 Expressed protein LOC_OsO3915920 Expressed protein LOC_0308908190 Uncharacterised protein family containing protein LOC_OsO6937070 Expressed protein LOC_OsO5933620 Expressed protein LOC_Os12907550 Expressed protein LOC_Os11905490 Expressed protein ' LOC_Os11928420 Expressed protein LOC_OsO1971540 Expressed protein LOC_0306940520 Cell wall hydrolytic enzymes have been consistently found to be related to abscission. As we expected, four such enzymes including mannan endo-1 ,4-beta-mannosidase, pectinesterase family protein, endo-beta-mannanase and glycosyl hydrolases family 16 protein were up-regulated by SM. These enzymes can degrade cell wall component during abscission process. Two expansin genes were induced by SM. Expansins are cell wall associated enzymes involved in cell elongation. 3 A total of 19 pathogenesis-related genes showed increased expression in sh4 transgenic plants. Five genes encode chitinases including four chitinases and one glycosyl hydrolase family 18 protein. These chitinases can degrade chitin, the major cell wall component of fungi. Two genes encoding pathogenesis-related protein (PR) Bet v I family protein. In addition, genes encoding cell wall hydrolytic enzyme inhibitors, such as pectinesterase inhibitor domain containing protein, 42 wwwwwwwwwww polygalacturonase inhibitor 1 precursor, xylanase inhibitor protein 1 precursor and xylanase inhibitor protein 2 precursor, were also up-regulated by SM. They inhibit the activity of hydrolytic enzymes secreted by fungi to degrade plant cell wall during their infection. Stress related genes were up-regulated by SM. These stress responsive genes are usually up-regulated by ABA and abiotic stress such as drought, salt, cold, and wound. The up-regulation of these groups of genes was not previously reported associated with the abscission processes. Genes encoding transcription factors, signal transduction, transport, protein biosynthesis or degradation, and metabolism were also induced by SM, indicating abscission is an active process involving many aspects of molecular regulation. A relatively large number of genes involved in photosynthesis were up—regulated in sh4 transgenic plants. Genes Down-regulated by sh4 In comparison to the number of up-regulated genes, fewer genes were down-regulated by SM (Table 5). It is noticeable that of 37 down-regulated genes, 11 encode DNA binding/transcription factors (Table 6). 43 Table 5. Number of genes down-regulated by SM Functional catefigory Stage 1 Stage 2 Stage 3 Cell wall hydrolysis 0 0 1 Stress responsive 0 1 0 Pathogenesis-related 0 1 0 DNA binding/transcription factor 2 9 1 Signal transduction 0 3 0 Metabolism 0 4 0 Transport 0 2 0 Others 0 11 0 Unknown 0 5 0 Table 6. Genes down-regulated by SM Category Annotation ID Stage DNA binding/transcription factor Myb-like DNA-binding domain containing protein LOC_OsO1965370 2 Helix-turn-helix family protein LOC_OsO6939240 3 Helix-Ioop-helix DNA-binding domain containing protein LOC_OsO4923550 1,2 Helix—loop-helix DNA-binding domain containing protein LOC_OsO3953020 2 Transcription initiation factor MD, 31 kD subunit family protein LOC_OsO79421 50 2 AP2 domain containing protein LOC_OsO9g35010 1 AP2 domain containing protein LOC_OsO2945450 2 AP2 domain containing protein LOC_OsO3909170 2 Zinc finger, C2H2 type family protein LOC_OsO3g60570 2 Homeobox domain containing protein LOC_OsO4g45810 2 ijC domain containing protein LOC_OsO2gO1940 2 Metabolism Chemocyanin precursor, putative LOC_OsO3950160 2 Fatty acid elongase, putative LOC_0502911070 2 Phenylalanine ammonia-lyase, putative LOC_OsO2g41670 2 Oxidoreductase, 2OG-Fe oxygenase family protein LOC_OsO4949210 2 44 Table 6 cont’d Cell wall hydrolysis Beta-fructofuranosidase 1 precursor, putative Signal transduction Protein kinase domain containing protein Caltractin, putative Serine/threonine-protein kinase SAPK2, putative Transport Sugar transporter family protein Amino acid transporter, putative Others Heavy metal-associated domain containing protein ATPase, AAA family protein Reticulon family protein Harpin-induced protein 1 containing protein VQ motif family protein ZIM motif family protein Las1-like family protein Senescence-associated protein Hevamine A precursor, putative Ankyrin-1, putative AN1-like Zinc finger family protein Unknown Expressed protein Expressed protein Expressed protein Expressed protein Expressed protein ABA Regulation of Abscission Flower Shattering in Response of ABA Treatment LOC_OsO2901590 LOC_OsO1950410 LOC_OsO7g42660 LOC_OsO7942940 LOC_Os07939350 LOC_Os12908130 LOC_OsO4939360 LOC_Os12g28137 LOC_OsO1963240 LOC_OsO1964470 LOC_OsO3947280 LOC_OsO7942370 LOC_Os12908800 LOC_OsO3g1 3840 LOC_OsO1964110 LOC_0508942960 LOC_OsO9g21 710 LOC_OsO1 903980 LOC_OsO1g72360 LOC_OsO3g1 3870 LOC_OsO3932420 LOC_OsO3932490 N MN NNNNNNNNNNN NNNNN Our microarray data suggested ABA and stress related genes were up-regulated by SM, which led to an investigation of the role of ABA in rice grain 45 abscission. Young panicles that just began to flower were treated with 0.1 mM and 1.0 mM ABA. Panicles treated with H20 were used as the control. Percentage of flower shattering was scored every 24 hr. Both 0.1 mM and 1.0 mM ABA caused flower abscission. After 24 hours in 1.0 mM ABA, nearly 60% of flowers shattered, and at 48 hr almost all flowers dropped when tapped by hand. In response of 0.1 mM ABA treatment, the percentage of flower shattering increased gradually with the length of treatment, and about 60% flowers shattered after 96 hr (Figure 8). 100- 1.0 mM § m 80* .E '5 60. 01mM 1:: . m .C U) 40‘ h (D E, 20~ LI. 0 , . . . 24 48 72 96 Hours in ABA Figure 8. O. nivara flower shattering in response of ABA treatment. Same developmental stage young panicles were treated with 0.1 mM and 1.0 mM ABA. Percentage of flower shattering were scored every 24 hrs. Each dot represents the average plus standard deviation of three replicates. Panicles from T2 transgenic plants with and without the trangene were treated with 1.0 mM ABA under the same conditions. For plants with the transgene, The 46 average force required to pull away flowers from the pedicels became substantially lower than that treated with H2O after 48 hours of the treatment. Wherease plants that did not carry the SM transgene showed no difference between the ABA and H20 treatment (Figure 9). 240- 2001 -I« .1. 160 - 120 j 801 40- Force (9) H20 ABA Figure 9. Flower shattering of T2 transgenic plants and the control segregated from the same T0 parent S12 in response of ABA treatment. Force required to pull away flowers from the pedicels was measured at 48 hours after the treatment. Black bar indicates plants with the SM transgene white bar indicates plants without the transgene. Evaluation of Seed ABA Content at Different Developmental Stages Our in vitro experiment demonstrated that ABA could trigger abscission of rice flowers that were either pollinated or not pollinated. Because ABA was found accumulate in developing embryos for the preparation of seed desiccation and dormancy, we tested the hypothesis that ABA synthesized in the developing embryos serves as the source of abscission signal. To test the hypothesis, we 47 removed stigmas of unopened flowers of the wild species 0. nivara so that embryo development was blocked. These flowers stayed on the panicle while grains with mature seeds shattered. This suggested that signals from developing rice embryo were required for grain shattering. We measured ABA content of the developing seeds of O. nivara. After anthesis, ABA content per seed increased substantially, especially between 6 to 9 days after pollination. ABA content kept increasing and reached the highest level at 12 days after pollination (Figure 10). This was positively correlated with the expression of SM and negatively correlated with the force required to break away the grain from the pedicel (Li et al. 2006b). 4.0- , ‘5 . ~ E 30 e 'O 3 2.o~ & 2 m S. 1.04 0 fl . . . 6 9 12 15 Days after pollination Figure 10. Seed ABA content in O. nivara. Seed ABA was measured at 6, 9, 12 and 15 days after pollination. 48 DISCUSSION Microarray Analysis With full genome sequences available, microarray analysis has become one of the most powerful tools to analyze the profiles of gene expression at the genome-wide scale. This method has provided insights into gene function and interactions (Brown and Botstein, 1999; Young 2000; Lockhart and Winzeler, 2000). To deal with such a large amount of data, it is critical to employ statistical methods to reduce the error rate and bias. Although several statistical methods have been recommended for the data analysis, there has been no standard method that works best for every dataset (Clarke and Zhu, 2006). A popular pre-processing step for two-color array is background correction prior to normalization. The goal of background correction is to correct the foreground intensities for background noise within the spotted region. The most common approach is “Subtract” background correction, which approximates the background at each spot by measuring the local, off-spot signal intensity and subtracting the value from the foreground signal. The “Minimum” or “Half” method subtracts local, off-spot signal intensity from each spot’s foreground intensity unless the net value is non-positive. In the latter case, the net intensity is set to 0.5 in “Half” strategy, and one-half the minimum positive net intensity in “Minimum” method (Smyth and Speed, 2003). 49 I compared several background adjustment options available in the Limma package. The aberrant log-ratio M and log-intensity A plot (MA-plot) after “Subtract”, “Minimum” or “Half” background corrections implied that these adjustments were not suitable for the dataset (Figure 7). This may be explained in two ways. Firstly, because the portion of the slide used to estimate background does not have any DNA bound, we are actually estimating the background binding of the spot by using a portion of the slide that might not be very similar (Zhang et al., 2006). Secondly, the simple “Subtract” method excluded the negative background-corrected intensities and lost differential expression information for weakly expressed genes, while spots with small positive intensities corrected by “Minimum” and “Half” were usually unreliable estimates of M and A. These spots had substantially variable log-ratios that challenged the downstream analyses, and might have lost differential expression information of these genes (Zhang et al., 2006). From our MA-plot, it is obvious that a large number of genes were weakly expressed or not expressed at all. Therefore, subtraction based background corrections were not suitable for our dataset. Although the RMA method yielded better MA-plot than subtract based background correction methods, it was still biased for our dataset, especially for some weakly and moderately expressed genes. The assumption of RMA (Bolstad 2004) is that the observed intensity is composed of the true intensity which is exponentially distributed (and hence positive) and the random noise 50 which is normally distributed (maybe truncated at zero). Thus this model does not return negative intensity values, which is better than the conventional subtract background correction. While some studies applied background correction in their microarray analyses, others have proposed ignoring the information in the background intensities (Parmigiani et al., 2003; Qin et al., 2004). Whether to apply background adjustment or not should depend on individual dataset. Prior to using any normalization options, we should always compare the MA-plot before and after various background corrections and determine whether background adjustment is appropriate. In many cases background correction can remove systematic bias, but it is better not to use background correction if the background intensity is low and relatively consistent across arrays. Gene Exgression Regulated by SM Two categories of genes identified in the present study were previously shown to be associated with abscission. The first category included genes encoding cell wall hydrolytic enzymes and expansins. Dissolution of the middle lamella or even primary cell wall is an essential step in the abscission process. The major components of the cell wall are pectins, cellulose, and hemicellulose (Carpita and Gibeaut, 1993; Chun and Huber, 1998). Abscission involves expression, synthesis and secretion of hydrolytic enzymes. 0f the enzymes we identified, pectinesterases are involved in pectin modification and make the wall more susceptible to other hydrolases (Brown 1997). It is interesting that the genes encoding cell wall hydrolytic enzymes were up—regulated by SM at the first developmental stage, which is the pollination stage. This suggested that these enzymes were synthesized well before grain abscission occurred. We also identified that two expansin genes were up-regulated by SM. Expansins were originally found to be related to cell elongation and elasticity, the up-regulation at the abscission zone suggested they are associated with abscission (Cho and Cosgrove, 2000; Belfield et al. 2005). Of various hydrolytic enzymes commonly associated with abscission, genes encoding polygalacturonase (PG) did not exhibit increased expression in sh4 transgenic plants. Because we were unable to obtain good-quality RNA at the developmental stage very near grain abscission, we cannot rule out the possibility that these genes are expressed at a later stage than what we examined. This hypothesis supported previous finding that the up-regulation of PG was only detected at the time of cell separation (Taylor et al., 1991; Bonghi et al., 1992; Hong and Tucker, 1998), although other studies suggested PG also exhibited increased expression prior to the onset of abscission (Kalaitzis et al., 1997; Gonzalez-Carranza et al., 2002). The second category is pathogenesis-related genes. The shedding of an organ provides a vulnerable site for pathogen invasion. It is therefore plausible for 52 pathogen related genes to be up-regulated during abscission process (Roberts et al., 2002). Indeed, defense genes were definitely associated with abscission (reviewed by Patterson, 2001; Roberts et al., 2002). Genes encoding chitinases and beta-1,3-glucanases have been observed to be up-regulated in the abscission zone (Volko et al., 1998; Kubigsteltig et al., 1999). Of the 19 defense related genes, 15 of them were up-regulated by SM atthe first developmental stage, suggesting they may play a role in induced resistance of fungal attack after a grain is shed. In addition to the two categories of abscission related genes found in previous studies, we have identified several other functional groups of genes regulated by SM, such as genes associated with metabolism, signal transduction, transcription factors, membrane, transport, protein biosynthesis and modification, and photosynthesis. Abscission is an active biological process. Previous studies have concluded that RNA and protein synthesis is very active prior to cell separation (Abeles 1968; Abeles and Leather, 1971; Addicott 1982). Thus we expect to see dynamic biochemical activities during the abscission process. Given the hypothesis that cell wall hydrolytic enzymes were synthesized well prior to the onset of abscission, it makes sense that genes involved in protein biosynthesis and modification were up-regulated. Initiating abscission requires perception of environmental or hormone signals and the signal transduction messengers. It is noticeable that sh4 regulated a 53 number of transcription factors. As a transcription factor itself, sh4 may function relatively upstream in the abscission pathway. We should keep in mind that rice grain abscission zone is composed of mostly one layer of cells. However, tissue sample for the microarray study is composed of many more layers of cells at both sides. Thus, not all candidate genes identified in this analysis are abscission specific. Recently optimized laser capture microdissection (LCM) methods have been successfully used by the Lashbrook lab at Iowa State University to isolate highly enriched populations of stamen abscission zone cells from paraffin-embedded flowers in Arabidopsis. If this method works in rice, abscission-layer specific mRNA may be obtained for future studies. ABA Regulation of Abscission Another large category of genes is ABA and/or stress related genes. Most of these genes are Inducible by ABA and/or abiotic stresses, such as drought, wound, salt, and cold stresses. Two genes encoding late embryogenesis abundant (LEA) proteins were also up-regulated by SM. The elevated expression of LEA has been observed under both ABA and stress treatment in rice (Rabbani et al., 2003). These proteins act as a desiccation protectant and are involved in protecting macromolecules such as enzymes and lipids (Moons et al., 1997). It has been well documented that ABA is accumulated under abiotic stresses and plays an 54 important role in the plant response to abiotic stresses (Ingram and Bartels, 1996; Shinozaki and Yamaguchi-Shinozaki, 2000; Zhu 2002). It has also been proposed that stress responsive genes are up-regulated by both ABA-dependent and ABA—independent signaling pathways (Shinozaki and Yamaguchi-Shinozaki, 2000; Zhu 2002). SM can induce the expression of ABA and stress responsive genes when plants were not subject to stresses, suggesting it interacts with ABA signaling pathway. This finding was further confirmed by the subsequent ABA experiments. Our results indicated that application of exogenous ABA was capable of promoting rice flower abscission, but only in the wild species 0. nivara or in sh4 transgenic cultivar. This suggests that the abscission accelerating activity of ABA requires the participation of sh4 of the wild species. Our microarray analysis opens a door to further investigate the function of genes involved in abscission as well as a network within and across the abscission related metabolic pathways. We demonstrated that ABA is the plant hormone that triggers rice grain abscission. This contrasts with many other studies that emphasized the essential role of ethylene in signaling abscission. Our results support that dicotyledons and monocotyledons may have differentiated in hormone signaling for organ abscission (Sargent et al., 1981, 1984). More importantly, this research established the first connection between ABA and a gene regulating plant organ abscission. This sets the stage for future studying hormone signaling network and molecular developmental pathways associated 55 with organ abscission in rice and other grasses, which remain totally unknown to date. 56 APPENDIX Author’s Publication List During the Master’s Program Li C, Zhou A, Sang T (2006) Rice domestication by reducing shattering. 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