ABSTRACT
    Citrus crop represents the highest fruit production in the world. The production in 2008 was more than 100 million tones (www.fao.org). Spain is one of the main producer countries in the world and the principal exporting country of citrus fruits for fresh consumption. Therefore, citrus crop constitutes an important source of wealth for the worldwide and national agricultural sector. The final production depends on physiological factors inherent to fruit development and its ability to resist adverse environmental conditions and remain attached to the main body of the plant, overcoming the abscission. The current knowledge of abscission at the molecular level is limited if it is compared with the large volume of data related to other physiological processes. In order to extend this knowledge, this work focuses on the global analysis of the anatomical, transcriptomic and biochemical changes during citrus fruit abscission.
    The first approach carried out was the anatomical characterization of the fruit abscission zone C (AZ-C) during the activation of the process. For this purpose, abscission kinetics was established in two varieties of sweet orange [Citrus sinensis (L.) Osb. cv. ‘Washington Navel’ and cv. ‘Navel Ricalate’] under in vitro ethylene and 1-aminocyclopropane-1-carboxylic acid treatments. Based on abscission kinetics a temporal sequence of samples was determined to study the abscission zone anatomy using different microscopy and histochemical techniques. The anatomical study of the AZ-C allowed to define the specific changes that occur during the activation of the fruit abscission at the cellular level and determine exactly the number of cell layers that constitute the AZ-C. This fact made possible to isolate specific cells from this tissue for further work. 
    The analysis of the transcriptomic changes brought about during abscission was achieved using the Spanish Citrus Functional Genomics Project (CFGP) cDNA microarray (Martínez-Godoy et al., 2008) that contains 21.081 unigenes. The microarray was hybridized with samples from the AZ-C and fruit rind that were isolated using laser capture microdissection. The temporal sequence of samples was determined after the establishment of the abscission kinetics of ethylene-treated fruits. By comparing the associated gene expression for each tissue during the ethylene treatment, genes or groups of genes with a potential role in the abscission process were determined. 
    The results of the expression analysis suggest a sequential action of the cellular machinery during the development of the abscission process. The AZ-C induces hormone and reactive oxygen species (ROS) mediated signaling pathways, a fact reflected in the activation of hormone response transcription factors and proteins involved in hormone synthesis and ROS detoxification. On the other hand, genes encoding kinases and receptor-like proteins are induced in the AZ-C. These proteins would be responsible of the transduction of abscission signals. Moreover, there is a protein turnover (synthesis and degradation) in response to the new metabolism and activity of the AZ-C cells. At the same time, the AZ-C activates transcription regulation by inducing transcription factors of different gene families (MADS-box, bHLH or MYB). On the other hand, the up-regulation of genes related to intracellular trafficking of vesicles would be associated with the redistribution of proteins in the AZ-C. Finally, the AZ-C activates the machinery of cell wall remodeling, which will enable the effective separation of the organ, and defense pathways against pathogens as prevention. In parallel, the expression results concerning the synthesis of lignin and cutin suggest that these polymers are synthesized and deposited in the AZ-C. These data were confirmed also by specific staining of the AZ-C and through the quantification of intermediates of the biosynthesis pathway in the AZ-C cells.
    The expression profile analysis in the AZ-C and the fruit rind of genes associated with the traffic of vesicles allowed to determine their putative function in the abscission process. This study revealed that AZ-C cells promote mainly the secretory pathway and reduce the activity of endocytic, vacuolar and recycling pathways. This traffic would be related to the extracellular contribution of cell wall modifying enzymes, that lead to cell separation during abscission, and lignin and cutin monomers that facilitate the mechanical breakdown of the AZ-C and seal the wound produced after abscission.
    An exhaustive analysis of the gene families related to cell wall modification allowed to identify, within the large number of proteins that normally constitute these families, those enzymes that could act specifically in the abscission process. For this purpose, a phylogenetic study was carried out. This analysis included the putative cell wall modifying enzymes identified in the version v0.9 of the assembly of the Citrus clementina haploid genome, ethylene-regulated proteins in AZ-C and fruit rind cells and abscission-related proteins from other plant species. On the other hand, an in silico differential gene expression analysis of the citrus genes from these families in different AZs as well as a promoter sequence analysis of some cell wall-related proteins involved in citrus fruit abscission was carried out. Moreover, this study was supplemented with an analysis of monosaccharide composition of the AZ-C cell walls using gas-liquid chromatography and with the immunolocalization of pectic polysaccharides of the AZ-C cell walls during the development of the abscission process. The results of this part of the work showed that changes in the organization of the cell wall polysaccharides are sufficient to reduce adhesion between cells and allow the effective separation of the fruit AZ-C. In addition, immunolocalization data correlated with the expression data of genes encoding enzymes that act on the pectic polysaccharides studied (pectin methylesterases, pectin acetylesterases, polygalacturonases, pectate lyases, ?-galactosidases and ?-xylosidases).