Asymmetry of physiological processes during abscission in tomato

Seminar I

Marko Chersicola

Mentor / Supervisor: prof. dr. Marina Dermastia

Odobril mentor / Approved by the supervisor: _____________________ (podpis/ signature)

Information and communication technologies 3

Doctoral degree

Ljubljana, 2013

Contents Abstract: ........................................................................................................................3 1. Introduction ..............................................................................................................3 2. Research objectives and methods ....................................................................................7 3. Discussion .................................................................................................................8 4. References ................................................................................................................9

Abstract: Abscission is a highly regulated process in which various organs, including leaves, flowers and fruits, are separated from the mother plant as a natural stage of plant development. Abscission occurs specifically in the abscission zone (AZ) tissue. AZ has a crucial role in the process. Not much is known about the molecular regulation of abscission. However, we have recently shown that the programmed cell death (PCD) is involved in the abscission of tomato leaves and flowers and suggested that various abscission related processes occur asymmetrically between the AZ proximal and distal sides. Our research is aimed to further characterize the identified asymmetry in the AZ and investigate its functional significance for the abscission process of flowers and leaves in tomato. We will investigate: (a) presumably high metabolic activity and membrane trafficking of cells at the proximal side of AZ, including the process of endoreduplication; (b) PCD type in AZ; (c) ethylene biosynthesis and perception; (d) cell wall metabolism/modification related gene expression and global transcriptomic changes at the late stage of abscission; and (e) if inhibition of PCD leads to inhibition of abscission. We will examine changes of expression of specific genes using quantitative RTPCR (qPCR), and use in situ hybridization and immunolocalization to determine in what AZ cells the gene transcripts and synthesized proteins are localized. Genes examined will be LX RNase, ACC oxidase (involved in ethylene biosynthesis), polygalacturonases, vacuolar protease with caspaselike activity. Ultrastructural changes in AZ of leaves and flowers will be further examined using transmission electron microscopy (TEM). For achievements of some goals the approaches of functional genomics will be applied. The results of the proposed research will supply information required for determining the functional significance of the observed asymmetry in the AZ and confirmation of the suggested hypotheses. The results could reveal novel and significant insights in three aspects: (1) the functional organization of the AZ in respect to the late execution stage of the process, and the significance of separating the execution machinery of abscission from the other involved factors (ethylene, LX RNase and PCD); (2) the role of a PCD process in ethylenerelated processes or in another, yet unclear, way in the execution mechanism of abscission; (3) the role of ethylene as a signal for inducing LX RNase and PCD in abscission. The findings of this study will contribute to the overall basic knowledge on the process of abscission that is still extremely rudimentary. Results will be directly applicable in agriculture and biotechnology for controlled abscission of fruits and manipulation of abscission time. Keywords: tomato, abscission, LX RNase, programmed cell death, ethylene

1. Introduction

Tomato as food Tomato is one of the most consumed vegetables in the world and is the dietary source of vitamins, minerals and fiber, which are important for human nutrition and health. Fresh fruits are used in salads, various culinary preparations, juices, or processed in the form of purees, concentrates, condiments and sauces. Tomato plants are grown worldwide in the field, or in greenhouses. Tomatoes rank fourth among the leading world vegetables. In 2001, over 100 million metric tons were produced, with the 15 leading countries being (in descending order) China, US, India, Turkey, Egypt, Italy, Spain, Brazil, Islamic Republic of Iran, Mexico, Greece, Russian Federation, Ukraine, Chile, and Uzbekistan (FAOSTAT 2011). Interestingly, the countries that produce higher yields do not possess the ideal climate for the tomato crop and have less land area devoted to tomato production. Northern European countries, as well as Canada and New Zealand, produce most of their tomatoes under controlled greenhouse conditions. Tomato consumption has also shown a general increased trend of consumption over a period of time. Tomatoes supply a mean of 12.1 kg/cap/yr, and tomato consumption is higher in Mediterranean and Arab countries (usually between 40 and 60 kg/cap/yr). Tomatoes are highly popular in Egypt, Italy, Israel, Lebanon, Turkey and United Arab Emirates (60-70 kg/cap/yr), whereas people from Greece and Libya have the highest preference consuming more than 100 kg of tomatoes per capita and year. Tomatoes are also a popular food in Latin and North America. Tomato is a rich source of nutrients (Table 1). Fresh tomatoes and tomato juices are high in water and low in calories. Both are good sources of vitamins A and C, but unfortified tomato juice has only about 2-3 the vitamin C content of raw, ripe (red) tomatoes. Similarly, canned tomatoes contain only about 3-4 times the vitamin C content of fresh ripe tomatoes. Ripe tomatoes contain 3-4 times the vitamin A as mature green tomatoes, but otherwise red and green tomatoes are about equal in nutritional value (Ensminger et al., 1995). Tomato puree and plain types of tomato sauce (without added ingredients such as meat or mushrooms) have about twice the solids content and about double the nutritional value of fresh tomatoes and tomato juice. Tomato popularity and its high level of consumption make this vegetable one of the major sources of vitamins and minerals in human diet (Peralta and Spooner 2007).

Domestication and taxonomy of tomato

There have long existed controversies regarding the place of domestication, early history, and taxonomy of tomato. The wild tomato species are native to western South America, from Ecuador south to northern Chile, and the Galapagos Islands. The putative progenitor of the cultivated species (Solanum lycopersicum = Lycopersicon esculentum var. cerasiforme) currently is widespread throughout warm regions of the world, but many of these are recent introductions. There are two competing hypotheses of the place of domestication of tomato, one supporting Peru, another in Mexico. While the Mexican origin is reasonable, we cannot discount a Peruvian origin, or even parallel domestication in both areas. Tomatoes were first recorded outside the Americas in Italy in 1544. They were cultivated first as ornamental or curiosity plants and thought by many to be poisonous. It was first accepted as a vegetable crop in southern Europe during the late sixteenth century. The first European cultivars had yellow to red flattened fruits, with deep furrows, and flowers with stigmas exserted from the anther tube. Derived cultivars had a wider range of fruit colors and shapes, smoother fruits, and stigmas included in the anther tube that led to increased fruit set but reduced the genetic variation of the crop. The taxonomy of tomato always has been controversial. This controversy involves not only generic placement in Lycopersicum or Solanum, but also hypotheses on interspecific relationships. Recent molecular data support treatment of tomato in Solanum (as we treat it here), and support allogamy, self-incompatibility, and green fruits as primitive of tomatoes. Tomato also serves as a model organism to understand the basic genetics of diploid plants. Features that enhance the usefulness of tomatoes for genetic studies are: the naturally occurring variability in the species, self-pollination that lead to the expression of recessive mutations, the possibility of controlled hybridization within and among species, the lack of gene duplication, and the possibility to easily identify the 12 chromosomes (Peralta and Spooner, 2007). In addition, the tomato belongs to the extremely large family Solanaceae and is closely related to many commercially important plants such as potato, eggplant, peppers, tobacco, and petunias. Knowledge obtained from studies conducted on tomato can be easily applied to these plants, which makes tomato important research material. Because of these facts, tomato serves as a model organism for the family Solanaceae and, specifically, for fleshy-fruited plants (Kimura et. al, 2008). Abscission Abscission is a natural part of plant development in which leaves, flowers or fruits, separate from the plant in a highly temporally and spatially regulated way (Roberts et al., 2002; Leslie et al., 2007). The basis for organ abscission is considered to be a cell separation process which occurs specifically in the preformed abscission zone (AZ) tissue located at the base of the organ to be shed. The abscission zone is morphologically and physiologically distinct from neighboring cells and includes smaller sized, cytoplasmically dense cells forming from 150 cell layers in different plants (Osborne and Sargent, 1976; Roberts et al., 1984; Leslie et al., 2007; van Nocker, 2009). In some plants there is more than one AZ tissue such as for the tomato fruit in which one AZ is localized between the fruit itself and the pedicel and a second AZ localized between the pedicel and the stem generating a joint. Although not much is known about the molecular regulation of abscission, based on knowledge accumulated from anatomical, physiological, genetic and molecular studies, the abscission process is suggested to require four successive phases for its successful execution (Roberts et al., 2000; Patterson, 2001; Taylor and Whitelaw, 2001; Jervis et al., 2003; Leslie et al., 2007). In recent years, progress has been made in the study of several phases of the abscission process largely in Arabidopsis, tomato and citrus model systems. Several proteins have been shown to have a regulatory role in abscission, including potential signal molecules and receptors (Lewis et al., 2006; Cho et al., 2008; McKim et al., 2008; Stenvik et al., 2008; Liljegren et al., 2009; van Nocker, 2009). Transcriptomic analyses during abscission have identified genes that are regulated during this process (Cai and Lashbrook, 2008; Agusti et al., 2009; Meir et al., 2010). In many cases, the abscission process is induced by ethylene, while the rate and degree of abscission depends upon the endogenous balance between auxin and ethylene levels in the tissue (Patterson, 2001; Taylor and Whitelaw, 2001; Roberts et al., 2002; Meir et al., 2006, 2010): auxin concentrations must be reduced in the AZ for rendering its sensitivity to ethylene, which promotes the advancement of abscission (Abeles and Rubinstein, 1964; Sexton and Roberts, 1982). An important step in ethylene biosynthesis is ACC oxidase that generates ethylene from 1aminocyclopropane1carboxylate (ACC) (Dorling and McManus, 2012). Based on their expression pattern and modulation in transgenic plants, genes encoding polygalacturonases (PGs) and β1,4glucanases (cellulases) have been suggested to play a central role in the execution of cell separation (Greenberg et al., 1975; Lashbrook et al., 1998; Brummell et al., 1999; Hong et al., 2000; GonzalezCarranza et al., 2007; Jiang et al., 2008; Ogawa et al., 2009). Other proteins associated with the AZ are expansin (Cho and Cosgrove, 2000; Belfield et al., 2005), pathogenesis related (PR) proteins (Eyal et al., 1993; Coupe et al., 1997) and metallothioneins (Coupe et al., 1995), but their role in the process is not yet clear.

We have recently shown (BarDror et al., 2011) that AZs of tomato leaf or flower are structured in an asymmetric manner after the abscission is triggered. Thus, cells comprising the proximal side of AZ, which are retained on the mother plant after the execution of abscission, differ substantially from those in the distal side that are going to be shed, in their morphology, biochemistry and metabolism. While the main process occurring in cells at the distal side is programmed cell death (PCD), cells at the proximal one exhibit several features presumably related to the extensive membrane trafficking and /or high metabolic activity. PCD – Programmed Cell Death PCD is a genetically encoded, active process leading to cell death. It is crucial to the development and survival of plants and is developmentally and environmentally regulated (Gunawardena, 2008; Rogers, 2005; van Doorn and Woltering, 2005). Cell death mechanisms in plants have been suggested to be subdivided into two classes of PCD: autolytic and nonautolytic that are analogous to autophagic PCD and necrosis in animals, respectively (van Doorn, 2011). In autolytic PCD, autophagosomes develop that may be detected using autofluorescent amine dye, monodansylcadaverine (MDC) (Contento et al., 2005). PCD is involved in plant embryogenesis, selfincompatibility, xylogenesis and senescence, as well as in the response to biotic or abiotic stresses (Fukuda, 2004; Lam, 2004; Bozhkov et al., 2005; Van Breusegem and Dat, 2006). In many plants ethylene signaling is employed during activation of different PCD processes (Trobacher, 2009). Ethylene signaling is both spatially and temporally regulated, which enables selective induction of PCD in the targeted tissues or cells. Death pathways are not conserved between animals and plants but seem to share common features (Podrabsky and Krumschnabel, 2010). Apoptotic-like features in plants’ PCD include for example mitochondrial role; the metacaspases; caspaselike activities in plant PCD; and functionality of the Bax Inhibitor1, a cell death suppressor (Watanabe and Lam, 2009). Few studies demonstrated functionality of nonplant apoptotic inhibitors following their heterologous overexpression in plants to modulate plant PCD (Li et al., 2010; Danon et al., 2004; Thomas et al., 2006; Lincoln et al., 2002). Hallmarks of PCD have been observed in distal side of AZ in tomato leaves that included loss of cell viability, altered nuclear morphology, DNA fragmentation, elevated levels of reactive oxygen species, and elevated enzymatic activities and expression of PCD associated genes. Overexpression of antiapoptotic proteins resulted in retardation of flower abscission, indicating PCD requirement (BarDror et al., 2011). Tomato LX RNase Previously, has been observed that a delay of tomato leaf abscission occurs with inhibited LX ribonuclease (Lers et al., 2006). The tomato LX RNase is a member of the T2/Slike RNases(Loffler et al., 1992). LX is accumulated in the ER and its C terminal amino acid sequence, HDEF was demonstrated to function as an ER retention signal (Lehmann et al., 2001; Kaletta et al., 1998). Expression of the LX gene is highly induced during leaf and petal senescence but can be induced also in young leaves by ethylene (Lers et al., 1998). The involvement of LX in other PCD processes was suggested by its induced expression during seeds germination and xylogenesis (Lehmann et al., 2001). Specific induction of the LX protein has been detected in the distal side of the mature tomato's AZ tissue and was inactivated by either pretreatment with the ethylene action inhibitor, 1methylcyclopropene (1MCP), or by application of auxin to the pedicel (BarDror et al., 2011). Endoreduplication It has been suggested that metabolically active or highly specialized cells may exit the classical cell cycle and enter the onset of endoreduplication cycle (Joubes and Chevalier, 2000; Dermastia, 2009). Endoreduplication is a variant of cell cycle, where nuclear DNA is replicated without subsequent mitosis, leading to endopolyploid cells. It is an important part of developmental processes such as cell fate maintenance (De Veylder et al., 2011). In plants, endoreduplication is widespread and is found in economically important tissues, such as cereal endosperm, cotton trichomes, tomato fruits and nitrogenfixing symbiotic root nodules in legumes (De Veylder et al., 2011). In storage endosperm tissue of maize, sorghum and teosinte, endoreduplication is highest in the largest cells in the central part of endosperm (Vilhar et al., 2002; Kladnik et al., 2006; Dermastia et al., 2009). In tomato, all organs are endopolyploid, including stem and leaf petiole, where nuclear DNA amount may reach 128C (1C is the size of an unreplicated haploid genome). Comparison of diploid and tetraploid plants revealed similar amounts of endopolyploid cells, indicating that endoreduplication is developmentally regulated (Smulders et al., 1994). It has been suggested that ethylene and auxin concentrations have an effect on endoreduplication (Gendreau et al., 1999; Ishida et al., 2010).

2. Research objectives and methods Previous results (BarDror et al., 2011) clearly demonstrate an asymmetry between the distal and proximal sides of the AZ in respect to the occurrence of PCD, expression of LX and localization of LX protein. We hypothesize that the enzymatic machinery required for execution of cell separation is produced in the proximal side of the AZ, while ethylene biosynthesis, LX and PCD induction occur mainly in the distal side. In respect to the specific induction of PCD and LX in the distal side, we will evaluate two hypotheses: a) LX function is required for PCD process to occur which in turn is needed for execution of abscission; b) LX has a regulatory role in modulating the ethylene biosynthesis or perception pathways and the induction of hormone level during abscission. Experiments will be performed to further characterize differences between the proximal and distal sides forming asymmetry in the AZ during different stages of the abscission process. Aspects to be characterized include: (1) PCD type and occurrence, ethylene biosynthesis and perception, cell wall metabolism/modification related gene expression and global transcriptomic changes; (2) to investigate the consequences of efficient LX suppression to abscission, PCD and the observed asymmetry in the AZ; and (3) to examine if inhibition of PCD will lead to inhibition of abscission. We will examine changes of expression of specific genes using quantitative RTPCR (qPCR), and use in situ hybridization and immunolocalization to determine in which AZ cells the gene transcripts and synthesized proteins are localized. Ultrastructural changes in AZ of leaves and flowers will be further examined using transmission electron microscopy (TEM). METHODS

a) Estimation of nuclear DNA endoreduplication in cells of proximal and distal side of AZ by image cytometry

Histological sections will be stained by Feulgen reaction that stoichiometrically stains DNA. The quantity of DNA in the nuclei will be measured by densitometric computer-based image cytometry. The image analysis system grabs images from the microscope via a digital camera, and calculates absorbance from the grey values of pixels in the nucleus (Vilhar and Dermastia 2001; Vilhar et al. 2001; Kladnik et al. 2004a; Kladnik et al. 2006). We will use the interphase peak method adapted for use with tissue sections established in our lab, which allows spatial and temporal examination of the distribution of nuclei (Vilhar et al., 2001, 2002; Vilhar and Dermastia, 2002; Dermastia et al., 2009). Tomato (Solanum lycopersicum cv. VF36) plants will be grown either in the greenhouse or in growth chambers (BarDror et al., 2011). Abscission experiments will be performed in planta as described (Lers et al., 2006; Meir et al., 2006, 2010). For induction of leaf abscission the leaf blades of up to four bottom leaves will be removed with a sharp razor, leaving most of the petioles intact. For abscission acceleration, ethylene will be applied 36 h later to the debladed tomato plants by placing them for 24 h in sealed containers with ethylene atmosphere. Endoreduplication will be estimated on the histological slides prepared from the AZs of plants at different times after start of ethylene induction. Part of the stem containing AZ will be excised from control or abscission induced plants and fixed in FAA (formalin, acetic acid, ethanol). Median longitudinal sections will be cut with a microtome to obtain a longitudinal view of the AZ. Hipothesis: - Cells with highly endoreduplicated nuclear DNA are limited to the proximal side of AZ where intensive membrane trafficking has been detected. - Nuclei with higher levels of endoreduplication are previously observed big ameboidal ones (Dermastia et al.)

b) IMMUNOLOCALISATION/IN-SITU HIBRIDISATION/TUNEL ASSAY Plant tissues in different phases of AZ development will be fixed in suitable fixatives and sectioned on a microtome. Immunohistochemical localization analysis of plant vacuolar protease, ACC oxidase and polygalacturonases in AZ sections will be performed to determine the specific cells in which these proteins are expressed, utilizing fluorescence microscopy and fluorescently labeled secondary antibodies (commercially available or custom made) (BarDror et al., 2011). Changes in nucleus morphology will be examined by staining DNA with fluorochrome DAPI (4,6 diamidino2phenylindole). DNA fragmentation will be followed using the TUNEL assay (TdTmediated dUTP nick end labeling) as described (Kladnik et al., 2004b; Kladnik et al., 2005; BarDror et al., 2011) using a commercially available kit. Autophagosome development will be examined by staining of fresh tissue sections with a fluorescent dye

monodansylcadaverine (Contento et al., 2005). The expression of LX in the AZ will be examined with in situ hybridization of LX mRNA in tissue sections, using digoxigenin labeled oligonucleotide probes and alkaline phosphatase labeled antiDig antibodies as previously described (Kogovšek et al., 2011). Quantitative realtime RTPCR will be used for quantification of selected gene expressions using Oligo dT primers designed by using available software (BarDror et al., 2011). To analyze gene expression during leaf abscission, the AZs will be separated into proximal and distal parts and thin layers of tissue will be excised from each using a scalpel and immediately frozen in liquid nitrogen. RNA will be extracted and cDNA will be synthesized. Leaf AZ samples will be prepared as in WP1. In addition, in experiments designed to follow flower abscission, flowers at similar levels of development will be cut at their base with a sharp razor to induce pedicle abscission essentially as described (BarDror et al., 2011). A day before the experiment, all opened flowers will be removed from the plants and only flowers which opened the night before the initiation of the experiments will be used. Induced abscission experiments in flowers will be performed in planta. Molecular and biochemical studies in AZ WP2 is a part of larger study carried out together with the team of Dr. Lers from the Volcani Center, Israel in which hypotheses that (a) LX function is required for PCD process to occur which in turn is needed for execution of abscission and (b) LX has a regulatory role in modulating the ethylene biosynthesis or perception pathways and the induction of hormone level during abscission, are tested. Hipoteses: - Plant vacuolar protease with caspase-like activity is localized at the distal side of AZ where PCD occurs. - Ethylene biosynthesis occurs at the distal side of AZ, where LX is localized and where a positive TUNEL reaction was detected. - Polygalacturonases are located mostly at the proximal side of AZ. - AZ is structured asymmetrically in flowers and fruits and accordingly DNA damages related to PCD are visualized by positive TUNEL reaction. Autophagosomes develop in cells at the distal side of AZ - Expression of gene encoding LX is mostly at the distal side of AZ, where the LX protein is localized. STUDIES PERFORMED ON TRANSGENIC PLANTS:

i) Study of AZ in RNAi LXinhibited transgenic tomato lines A larger study carried out together with the team of Dr. Lers from the Volcani Center, Israel in which hypotheses that inhibition of PCD results in a lower level of ethylene biosynthesis and a delay in the progress of abscission will be investigated by functional genomics approaches. RNAi LXinhibited transgenic tomato lines will be produced at the Volcani Center and studied as described earlier. Plant tissue will be prepared essentially as described elsewhere (BarDror et al., 2011) and examined with a transmission electron microscope. Our tasks: - Ultrastructural analysis of AZ - TUNEL assay on AZs - Immunolocalization of plant vacuolar protease with caspaselike activity - Immunolocalization of ACC oxidase involved in ethylene biosynthesis - Immunolocalization of the polygalacturonases - In situ hybridization of LX

i) Study of AZ in plants overexpressing antiapoptotic proteins A larger study carried out together with the team of Dr. Lers from the Volcani Center, Israel in which the following hypotheses are tested: (a) that observed significantly delayed leaf and flower abscission in the transgenic plants overexpressing genes encoding for antiapoptotic proteins is associated with the observed PCD in the AZ and (b) that the normal progress of the observed PCD in the AZ is required for progress of the abscission process. Transgenic tomato lines overexpressing antiapoptotic proteins will be produced at the Volcani Center and studied as described earlier. Our tasks: - Ultrastructural analysis of AZ - TUNEL assay on AZs

3. Discussion

Abscission in plants is a highly temporally and spatially regulated process in which various organs, including leaves, flowers or fruits are detached from the main plant body. The occurrence of abscission has a large agricultural implication both on the level of commodities growth and during storage and shelf life of fresh produce. In different agricultural commodities there is a need to either inhibit or induce abscission. The premature induction of abscission in trees, flowers and very young fruits as a result of extreme temperature has a significant negative implication for yield and prices. Similarly, acceleration of abscission in different fresh produce during postharvest life and storage results in significant loss of quality as for example abscission of cherry tomato from the bunch (BenoMoualem et al., 2004) or inflorescence in ornamentals (van Doorn and Stead, 1997; van Doorn, 2002). To overcome the problem, many crops are treated after harvest with chemicals to delay or prevent abscission (van Doorn and Woltering, 1991). On the other hand, in other agricultural commodities it is the acceleration of the abscission process which is required at a given time which to be beneficial and allow improved mechanization and saving labor, for example defoliation of cotton leaves before boll harvest (Faircloth et al., 2004) or weakening of fruit trees flowers or young fruitlets abscission zone for thinning the fruit load (Link, 2000). The basis for organ abscission is considered to be a cell separation process which occurs specifically in the preformed abscission zone (AZ) tissue located at the base of the organ to be shed (Osborne and Sargent, 1976; Roberts et al., 1984; Leslie et al., 2007; van Nocker, 2009). Based on knowledge accumulated from anatomical, physiological, genetic and molecular studies, the abscission process is suggested to require four successive phases for its successful execution (Roberts et al., 2000; Patterson, 2001; Taylor and Whitelaw, 2001; Jarvis et al., 2003; Leslie et al., 2007). The first phase involves early differentiation of cells localized at the site of the future organ separation into a determined AZ. In the second phase, the differentiated AZ acquires competence to respond to the abscission signal(s), presumably via developmental and hormonal cues. The third phase is the ethylene mediated execution of abscission via a cell separation process, which involves a major induction of cell wall modifying and hydrolytic enzymes, causing degradation of the middle lamella between AZ cells to allow physical separation of the abscised organ from the mother plant. The last phase, which follows detachment, includes the development of a protective layer at the surface of the exposed tissue, creating a scar at the site of organ detachment on the plant body. Based on their expression pattern and modulation in transgenic plants, genes encoding polygalacturonases and ß1,4 glucanases (cellulases) have been suggested to play a central role in the execution of cell separation (Greenberg s sod., 1975; Lashbrook s sod., 1998; Brummell s sod., 1999; Roberts s sod., 2002). The late stage of abscission in tomato leaf and flower is associated with a programmed cell death (PCD) in which T2/Slike RNase LX and nuclease BFN1 are involved. However, PCD is characterized only for cells at the distal side of the AZ, while cells at the proximal one show features of high metabolic activity and intensive membrane trafficking (BarDror et al., 2011). It has been proposed that cell types undergoing differentiation, putatively related with specific metabolic properties may exit the classical cell cycle and enter the onset of endoreduplication cycle whereby cells undergo successive rounds of genome duplication without going through mitosis (Joubes and Chevalier, 2000; Dermastia, 2009; de Veylder et al., 2011). As a consequence, endoreduplication leads to increase of nuclear DNA content and to endopolyploid cells. Several lines of evidence implied that ethylene may exert an effect on endoreduplication (de Veylder et al., 2011). Currently it is unknown if endoreduplication is associated with highly metabolically active cells at the proximal side of AZ. It is also largely unknown what may be the molecular basis of the observed asymmetry of processes in AZ. Based on results published in a previous paper (BarDror et al., 2011) we hypothesize that the enzymatic machinery required for execution of cell separation is produced in the proximal side of the AZ, while ethylene biosynthesis, LX and PCD induction occur mainly in the distal side. In respect to the specific induction of PCD and LX in the distal side, our long term goal is to test two hypotheses: a) LX function is required for PCD process to occur which in turn is needed for execution of abscission; b) LX has a regulatory role in modulating the ethylene biosynthesis or perception pathways and the induction of hormone level during abscission. In the proposed research we will further characterize the identified asymmetry of the abscission process of flowers and leaves of tomato at different levels using various approaches in order to better understand a functional significance for the abscission process and the involved PCD.

4. References Abeles, F.B., and Rubinstein, B. (1964). Regulation of Ethylene Evolution and Leaf Abscission by Auxin. Plant Physiol 39, 963969.

Agusti, J., Merelo, P., Cercos, M., Tadeo, F.R., and Talon, M. (2009). Comparative transcriptional survey between lasermicrodissected cells from laminar abscission zone and petiolar cortical tissue during ethylenepromoted abscission in citrus leaves. BMC Plant Biol 9, 127. BarDror, T., Dermastia, M., Kladnik, A., Znidaric, M.T., Novak, M.P., Meir, S., Burd, S., PhilosophHadas, S., Ori, N., Sonego, L., Dickman, M.B., and Lers, A. (2011). Programmed Cell Death Occurs Asymmetrically during Abscission in Tomato. Plant Cell 23, 41464163. Belfield, E.J., Ruperti, B., Roberts, J.A., and McQueenMason, S. (2005). Changes in expansin activity and gene expression during ethylenepromoted leaflet abscission in Sambucus nigra. Journal Of Experimental Botany 56, 817823. BenoMoualem, D., Gusev, L., Dvir, O., Pesis, E., Meir, S., and Lichter, A. (2004). The effects of ethylene, methyljasmonate and 1MCP on abscission of cherry tomatoes from the bunch and expression of endo1,4betaglucanases. Plant Sci. 167, 499507. Bosse, D., and Kock, M. (1998). Influence of phosphate starvation on phosphohydrolases during development of tomato seedlings. Plant Cell Environ. 21, 325332. Bozhkov, P.V., Filonova, L.H., and Suarez, M.F. (2005). Programmed cell death in plant embryogenesis. In Current Topics in Developmental Biology, Vol 67, G.P. Schatten, ed (San Diego: Elsevier Academic Press Inc), pp. 135179. Brummell, D.A., Hall, B.D., and Bennett, A.B. (1999). Antisense suppression of tomato endo1,4betaglucanase Cel2 mRNA accumulation increases the force required to break fruit abscission zones but does not affect fruit softening. Plant Molecular Biology 40, 615622. Cai, S.Q., and Lashbrook, C.C. (2008). Stamen abscission zone transcriptome profiling reveals new candidates for abscission control: Enhanced retention of floral organs in Transgenic plants overexpressing Arabidopsis ZINC FINGER PROTEIN2. Plant Physiology 146, 13051321. Cho, H.T., and Cosgrove, D.J. (2000). Altered expression of expansin modulates leaf growth and pedicel abscission in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U. S. A. 97, 97839788. Cho, S.K., Larue, C.T., Chevalier, D., Wang, H.C., Jinn, T.L., Zhang, S.Q., and Walker, J.C. (2008). Regulation of floral organ abscission in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U. S. A. 105, 1562915634. Contento, A.L., Xiong, Y., and Bassham, D.C. (2005). Visualization of autophagy in Arabidopsis using the fluorescent dye monodansylcadaverine and a GFPAtATG8e fusion protein. Plant Journal 42, 598608. Coupe, S.A., Taylor, J.E., and Roberts, J.A. (1995). Characterization of an messengerRNA encoding a metallothioneinlike protein that accumulates during ethylenepromoted abscission of sambucusnigra L leaflets. Planta 197, 442447. Coupe, S.A., Taylor, J.E., and Roberts, J.A. (1997). Temporal and spatial expression of mRNAs encoding pathogenesisrelated proteins during ethylenepromoted leaflet abscission in Sambucus nigra. Plant Cell Environ. 20, 15171524. Danon, A., Rotari, V.I., Gordon, A., Mailhac, N., and Gallois, P. (2004). UltravioletC overexposure induces programmed cell death in Arabidopsis, which is mediated by caspaselike activities and which can be suppressed by caspase inhibitors, p35 and Defender against Apoptotic Death. J. Biol. Chem. 279, 779787. De Veylder, L., Larkin, J.C., and Schnittger, A. (2011). Molecular control and function of endoreplication in development and physiology. Trends In Plant Science 16, 624634. Dermastia, M. (2009). Endoreduplication in cereal plants. New plant physiology research, 177202. Dermastia, M., Kladnik, A., Dolenc Koce, J., and Chourey, P.S. (2009). A cellular study of teosinte Zea mays subsp. parviglumis (Poaceae) caryopsis development showing several processes conserved in maize. Am J Bot 96, 17981807. Dorling, S.J., and McManus, M.T. (2012). The Fate of ACC in Higher Plants. In Annual Plant Reviews Volume 44 (WileyBlackwell), pp. 83115. Ensminger, A.H., M.E. Ensminger, J.E. Konlande and J.R.K. Robson (1995). The concise encyclopedia of foods and nutrition. CRC Press, Boca Raton, Florida, 1178 pp. Eyal, Y., Meller, Y., Levyadun, S., and Fluhr, R. (1993). A basictype PR1 promoter directs ethylene responsivness, vascular abd abscission zonespecific expression. Plant Journal 4, 225234. Faircloth, J.C., Edmisten, K.L., Wells, R., and Stewart, A.M. (2004). Timing defoliation applications for maximum yields and optimum quality in cotton containing a fruiting gap. Crop Sci 44, 158164. FAOSTAT 2011: http://faostat3.fao.org/home/index.html#DOWNLOAD Fukuda, H. (2004). Signals that control plant vascular cell differentiation. Nature Reviews 5, 379391. Gendreau, E., Orbovic, V., Hofte, H., and Traas, J. (1999). Gibberellin and ethylene control endoreduplication levels in the Arabidopsis thaliana hypocotyl. Planta 209, 513516. GonzalezCarranza, Z.H., Elliott, K.A., and Roberts, J.A. (2007). Expression of polygalacturonases and evidence to support their role during cell separation processes in Arabidopsis thaliana. Journal Of Experimental Botany 58, 37193730. Greenberg, J., Goren, R., and Riov, J. (1975). Role of Cellulase and Polygalacturonase in Abscission of Young and Mature Shamouti Orange Fruits. Physiologia Plantarum 34, 17.

Gunawardena, A.H.L.A.N. (2008). Programmed cell death and tissue remodelling in plants. J Exp Bot 59, 445451. Hong, S.B., Sexton, R., and Tucker, M.L. (2000). Analysis of gene promoters for two tomato polygalacturonases expressed in abscission zones and the stigma. Plant Physiology 123, 869881. Ishida, T., Adachi, S., Yoshimura, M., Shimizu, K., Umeda, M., and Sugimoto, K. (2010). Auxin modulates the transition from the mitotic cycle to the endocycle in Arabidopsis. Development 137, 6371. Jarvis, M.C., Briggs, S.P.H., and Knox, J.P. (2003). Intercellular adhesion and cell separation in plants. Plant Cell Environ. 26, 977989. Jiang, C.Z., Lu, F., Imsabai, W., Meir, S., and Reid, M.S. (2008). Silencing polygalacturonase expression inhibits tomato petiole abscission. Journal Of Experimental Botany 59, 973979. Joubes, J., and Chevalier, C. (2000). Endoreduplication in higher plants. Plant Molecular Biology 43, 735745. Kaletta, K., Kunze, I., Kunze, G., and Kock, M. (1998). The peptide HDEF as a new retention signal is necessary and sufficient to direct proteins to the endoplasmic reticulum. Febs Lett 434, 377381. Kimura S., and Sinha N. (2008).Tomato (Solanum lycopersicum): A Model Fruit-Bearing Crop, Cold Spring Harb Protoc; doi:10.1101/pdb.emo105. Kladnik, A., Vilhar, B., Chourey, P.S., and Dermastia, M. (2004a). Sucrose synthase isozyme SUS1 in the maize root cap is preferentially localized in the endopolyploid outer cells. Can J Bot 82, 96103. Kladnik, A., Chamusco, K., Dermastia, M., and Chourey, P. (2004b). Evidence of programmed cell death in postphloem transport cells of the maternal pedicel tissue in developing caryopsis of maize. Plant Physiology 136, 35723581. Kladnik, A., Chamusco, K., Chourey, P.S., and Dermastia, M. (2005). In situ detection of programmed cell death in the maize caryopsis. Period Biol 107, 1116. Kladnik, A., Chourey, P.S., Pring, D.R., and Dermastia, M. (2006). Development of the endosperm of Sorghum bicolor during the endoreduplicationassociated growth phase. J Cereal Sci 43, 209215. Kock, M., Loffler, A., Abel, S., and Glund, K. (1995). cDNA structure and regulatory properties of a family of starvationinduced ribonucleases from tomato. Plant Mol Biol 27, 477485. Kogovsek, P., Kladnik, A., Mlakar, J., Znidaric, M.T., Dermastia, M., Ravnikar, M., and PompeNovak, M. (2011). Distribution of Potato virus Y in Potato Plant Organs, Tissues, and Cells. Phytopathology 101, 12921300. Lam, E. (2004). Controlled cell death, plant survival and development. Nat Rev Mol Cell Biol 5, 305315. Lashbrook, C.C., Giovannoni, J.J., Hall, B.D., Fischer, R.L., and Bennett, A.B. (1998). Methods in floral abscission. Plant Journal 13, 303310. Lehmann, K., Hause, B., Altmann, D., and Kock, M. (2001). Tomato ribonuclease LX with the functional endoplasmic reticulum retention motif HDEF is expressed during programmed cell death processes, including xylem differentiation, germination, and senescence. . Plant Physiology 127, 436449. Lers, A., Sonego, L., Green, P.J., and Burd, S. (2006). Suppression of LX ribonuclease in tomato results in a delay of leaf senescence and abscission. Plant Physiology 142, 710721. Lers, A., Khalchitski, A., Lomaniec, E., Burd, S., and Green, P.J. (1998). Senescenceinduced RNases in tomato. Plant Molecular Biology 36, 439449. Leslie, M.E., Lewis, M.W., and Liljegren, S.J. (2007). Organ abscission In Plant Cell Separation and Adhesion, J.A. Roberts and Z.H. GonzalezCarranza, eds (Oxford: Blackwell Publishing), pp. 106136. Lewis, M.W., Leslie, M.E., and Liljegren, S.J. (2006). Plant separation: 50 ways to leave your mother. Curr. Opin. Plant Biol. 9, 5965. Li, W., Kabbage, M., and Dickman, M.B. (2010). Transgenic expression of an insect inhibitor of apoptosis gene, SfIAP, confers abiotic and biotic stress tolerance and delays tomato fruit ripening. Physiological and Molecular Plant Pathology 74, 363375. Liljegren, S.J., Leslie, M.E., Darnielle, L., Lewis, M.W., Taylor, S.M., Luo, R.B., Geldner, N., Chory, J., Randazzo, P.A., Yanofsky, M.F., and Ecker, J.R. (2009). Regulation of membrane trafficking and organ separation by the NEVERSHED ARFGAP protein. Development 136, 19091918. Lincoln, J.E., Richael, C., Overduin, B., Smith, K., Bostock, R., and Gilchrist, D.G. (2002). Expression of the antiapoptotic baculovirus p35 gene in tomato blocks programmed cell death and provides broadspectrum resistance to disease. Proc. Natl. Acad. Sci. U. S. A. 99, 1521715221. Link, H. (2000). Significance of flower and fruit thinning on fruit quality. Plant Growth Regul 31, 1726. Loffler, A., Abel, S., Jost, W., Beintema, J.J., and Glund, K. (1992). Phosphateregulated induction of intracellular ribonucleases in cultured tomato (Lycopersicon esculentum) cells. Plant Physiology 98, 14721478. McKim, S.M., Stenvik, G.E., Butenko, M.A., Kristiansen, W., Cho, S.K., Hepworth, S.R., Aalen, R.B., and Haughn, G.W. (2008). The BLADEONPETIOLE genes are essential for abscission zone formation in Arabidopsis. Development 135, 15371546. Meir, S., Hunter, D.A., Chen, J.C., Halaly, V., and Reid, M.S. (2006). Molecular changes occurring during acquisition of abscission competence following auxin depletion in Mirabilis jalapa. Plant Physiology 141, 16041616.

Meir, S., PhilosophHadas, S., Sundaresan, S., Selvaraj, K.S.V., Burd, S., Ophir, R., Kochanek, B., Reid, M.S., Jiang, C.Z., and Lers, A. (2010). Microarray Analysis of the Abscission Related Transcriptome in the Tomato Flower Abscission Zone in Response to Auxin Depletion. Plant Physiology 154, 19291956. Ogawa, M., Kay, P., Wilson, S., and Swain, S.M. (2009). Arabidopsis dehiscence zone polygalacturonase1 (ADPG1), ADPG2, and QUARTET2 are polygalacturonases required for cell separation during reproductive development in Arabidopsis. Plant Cell 21, 216233. Osborne, D.J., and Sargent, J.A. (1976). Positional Differentiation of Abscission Zones during Development of Leaves of SambucusNigra and Response of Cells to Auxin and Ethylene. Planta 132, 197204. Patterson, S.E. (2001). Cutting loose. Abscission and dehiscence in Arabidopsis. Plant Physiology 126, 494500. Podrabsky, J.E., and Krumschnabel, G. (2010). Cell death beyond worms, flies and humans: unusual model systems for cell death research. Apoptosis 15, 243248. Peralta I. E. and Spooner D. M. (2007). History, Origin and Early Cultivation ofTomato (Solanaceae); Genetic Improvement of Solanaceous Crops; Vol. 2; p. 1-25; Science Publishers. Roberts, J.A., Schindler, C.B., and Tucker, G.A. (1984). Ethylene Promoted Tomato Flower Abscission and the Possible Involvement of an Inhibitor. Planta 160, 159163. Roberts, J.A., Elliott, K.A., and GonzalezCarranza, Z.H. (2002). Abscission, dehiscence, and other cell separation processes. Annu Rev Plant Biol 53, 131158. Roberts, J.A., Whitelaw, C.A., GonzalezCarranza, Z.H., and McManus, M.T. (2000). Cell separation processes in plants Models, mechanisms and manipulation. Ann. Bot. 86, 223235. Rogers, H.J. (2005). Cell death and organ development in plants. In Current Topics In Developmental Biology, Vol 71, pp. 225+. Sexton, R., and Roberts, J.A. (1982). Cell Biology of Abscission. Annu Rev Plant Phys 33, 133162. Smulders, M.J.M., Ruskortekaas, W., and Gilissen, L.J.W. (1994). Development of Polysomaty during Differentiation in Diploid and Tetraploid Tomato (LycopersiconEsculentum) Plants. Plant Sci. 97, 5360. Stenvik, G.E., Tandstad, N.M., Guo, Y., Shi, C.L., Kristiansen, W., Holmgren, A., Clark, S.E., Aalen, R.B., and Butenko, M.A. (2008). The EPIP peptide of INFLORESCENCE DEFICIENT IN ABSCISSION is sufficient to induce abscission in Arabidopsis through the receptorlike kinases HAESA and HAESALIKE2. Plant Cell 20, 18051817. Taylor, J.E., and Whitelaw, C.A. (2001). Signals in abscission. New Phytologist 151, 323339. Thomas, S.G., Huang, S.J., Li, S.T., Staiger, C.J., and FranklinTong, V.E. (2006). Actin depolymerization is sufficient to induce programmed cell death in selfincompatible pollen. J. Cell Biol. 174, 221229. Trobacher, C.P. (2009). Ethylene and programmed cell death in plants. Botany 87, 757769. Van Breusegem, F., and Dat, J.F. (2006). Reactive oxygen species in plant cell death. Plant Physiol 141, 384390. Van Doorn, W.G. (2002). Effect of ethylene on flower abscission: a survey. Ann. Bot. 89, 689693. Van Doorn, W.G. (2011). Classes of programmed cell death in plants, compared to those in animals. Journal Of Experimental Botany 62, 47494761. Van Doorn, W.G., and Woltering, E.J. (1991). Developments in the use of growth regulators for the maintenance of postharvest quality in cut flowers and potted plants. Acta Horticulturae 298, 195208. Van Doorn, W.G., and Stead, A.D. (1997). Abscission of flowers and floral parts. Journal Of Experimental Botany 48, 821837. Van Doorn, W.G., and Woltering, E.J. (2005). Many ways to exit? Cell death categories in plants. Trends In Plant Science 10, 117122. Van Nocker, S. (2009). Development of the abscission zone. Stewart Postharvest Review 5, doi: 10.2212/spr.2009.2211.2215. Vilhar, B., and Dermastia, M. (2002). Standardisation of instrumentation in plant DNA image cytometry. Acta Botanica Croatica 61, 1126. Vilhar, B., Greilhuber, J., Koce, J.D., Temsch, E.M., and Dermastia, M. (2001). Plant genome size measurement with DNA image cytometry. Ann. Bot. 87, 719728. Vilhar, B., Kladnik, A., Blejec, A., Chourey, P.S., and Dermastia, M. (2002). Cytometrical evidence that the loss of seed weight in the miniature1 seed mutant of maize is associated with reduced mitotic activity in the developing endosperm. Plant Physiology 129, 2330. Watanabe, N., and Lam, E. (2009). Bax Inhibitor1, a Conserved Cell Death Suppressor, Is a Key Molecular Switch Downstream from a Variety of Biotic and Abiotic Stress Signals in Plants. Int. J. Mol. Sci. 10, 31493167.