Search

The juvenile to adult switch is the most important post-embryonic transition. In woody plants the juvenile phase can last many years with a great economic impact. In Arabidopsis, the small RNAs miR156 and miR172 play a crucial but opposite role in the regulation of this process. miR156 maintains juvenility, negatively regulating SPLs genes, while miR172 promotes adult transition, targeting the floral repressors AP2-like transcription factors. In this work, peach (Prunus persica L. Batch) orthologs of Arabidopsis epigenetic and genetic factors involved in the juvenility to adult phase transition were studied. In peach, higher levels of ppa-miR156 were detected in seedlings, in vitro and extra vitro plants than in adult plants. Also, PpSPLs were more expressed in adult plants, confirming a possible role for the miR156-SPL pathway in promoting juvenile-like characteristics. ppa-miR172 expression level was low in seedlings and in vitro plants but an increase was observed in the adult donor plant, corresponding to lower expression of PpAP2-like genes. In Arabidopsis, flower induction is also promoted by activation of the FLOWERING LOCUS T (FT) gene. In peach leaf tissue, low levels of PpFT-like expression in rejuvenated plants and seedlings were detected. We propose that, in peach, conserved key genes present in herbaceous plants and woody species are involved in juvenile to adult and adult to juvenile-like phase transitions.

Self-incompatibility is one of the most important barriers adopted by many flowering plants to prevent inbreeding, thus maintaining diversity within species. The elucidation of this reproductive constraint is crucial in olive (Olea europaea L.) because it may help to improve breeding strategies and orchard management. The available literature reports that a gametophytic self-incompatibility (GSI) system might exist in olive, even though neither cytological nor molecular data were provided. A molecular approach failed to find S-RNase (S-locus ribonuclease) and SLF (S-locus F-box containing protein), the genes responsible for GSI. In this paper, cyto-histological observations and bio-molecular evidence is presented, supporting the presence in olive of a sporophytic self-incompatibility (SSI) system. The main genes known to play a crucial role in SSI are SRK (S-locus receptor kinase) and SLG (S-locus glycoprotein), the female determinants, and SCR (S-locus cysteine rich protein), the male determinant. OeSLG and OeSRK genes cloned by PCR with degenerated primers were shown to be differentially expressed in flower organs of self-compatible (cv Frantoio) and self-incompatible (cv Leccino) genotypes by means of Real-Time PCR analysis. Since the same approach failed to isolate SCR, a 454 pyrosequencing library derived from flowers at different developmental stages was screened. One of the sequences displaying a conserved amino acid structure in terms of cysteine residuals, named OeSCR-like, proved to be specifically expressed in anthers at blooming stages. The overall data so far collected support the existence of an SSI system in olive, at least in the tested genotypes. INTRODUCTION Olive (Olea europaea L.), originally native of the eastern Mediterranean region and now spread in many other temperate parts of the world, is one of the oldest agricultural crop trees and it has been in cultivation for thousands of years. In spite of its great cultural and economical importance, very few studies have been undertaken on reproductive barriers, especially from the molecular standpoint. Along with male-sterility (Villemur et al., 1984; Bernard et al., 2000), self-incompatibility (SI) is the most effective reproductive barrier in olive (Diaz et al., 2006), although no molecular evidence for the system has to date been reported. SI is one of the most important systems to prevent inbreeding and it is actually divided into three main categories: two systems of gametophytic self-incompatibility (GSI) and one of sporophytic self-incompatibility (SSI) (Takayama et al., 2005). The gametophytic and sporophytic self-incompatibility systems are characterized by the different genetic behaviour of the pollen: the pollen SI phenotype in GSI is determined post-meiotically by its own haploid genome, whereas in SSI Proc. Intl. Workshop on Floral Biology & S-Incompatibility in Fruit Species Eds.: Sargent et al. Acta Hort. 967, ISHS 2012 134 incompatibility is established pre-meiotically and controlled by the diploid genome of the parent (Takayama et al., 2005). Furthermore, the presence of a hitherto unknown SI system in olive cannot be ruled out, as reported in Ipomoea trifida (Convolvulaceae) and Senecio squalidus (Asteraceae), even though their regulatory pathways remain unknown (Hiscock et al., 2003; Tomita et al., 2004). Olive is presently classified as a GSI species based on morphological traits, such as wet-type stigma and bi-nucleate pollen, features usually found in model taxa showing GSI (e.g. Antirrhinum, Rosaceae and Solanaceae), although no molecular evidences have been provided. In spite of this, in this paper we propose a SSI system of the Brassicaceaetype in olive. To the best of our knowledge, this is the first report dealing with the isolation of genes putatively involved in pollen-pistil interactions and the characterization of their expression levels and patterns in pistils and anthers throughout bloom. MATERIALS AND METHODS Plant Material and Cytological Analyses Plant materials were collected from the experimental fields of the Perugia CNR (Umbria, Italy). In order to study the behaviour of pollen grains and pollen tubes, selfand cross-pollinated pistils of one self-compatible (Frantoio) and three self-incompatible (Leccino, Moraiolo, Dolce Agogia) cultivars were fixed in Carnoy solution, stained by means of a solution of 0.1% aniline blue in phosphate buffer and 50% glycerol and then visualized under epi-fluorescence microscope. To get statistical validation, a total of 34,000 pollen grains were analyzed and the germination rates recorded for each cultivar by considering six different classes: a) pollen grains that did not germinate; b) pollen grains that germinated on the papillae cells; c) pollen grains that germinated on the stigma surface; d) pollen tubes reaching the proximal part of the style; e) pollen tubes reaching the middle part of the style; f) pollen tubes reaching the distal part of the style, after both selfand cross-fertilization. Molecular Analyses Molecular analyses were performed using total RNA extracted from flowers of cultivars Leccino (self-incompatible) and Frantoio (self-compatible) at different developmental stages using the RNeasy Plant Mini Kit (Qiagen) and treated by DNAse (Qiagen) according to the manufacturer’s protocols. All the cDNA samples were produced from total RNA through a RT-PCR using the SuperScript VILO cDNA Synthesis Kit (Invitrogen) according to the manufacturer’s protocol. The female and male determinants for the GSI and SSI systems, typical of the Solanaceae and Brassicaceae, respectively, were searched by means of PCR using degenerate primers. S-RNase (S-locus RNase) and SLF/SFB (S-locus F-box protein) amplifications were initially attempted, then analyses were focused on SLG (S-locus glycoprotein), SRK (S-locus receptor kinase), and SCR (S-locus cysteine rich protein). Flower-specific 454 pyrosequencing libraries were screened for the identification of SCR-like sequences based on protein domains available on NCBI databases and putatively involved in pollen-pistil interactions (Schopfer et al., 1999; Vanoosthuyse et al., 2001; Mishima et al., 2003; Higashima, 2010). All amplicons were sub-cloned using the TOPO-TA Cloning Kit (Invitrogen) according to the manufacturer’s protocol. All PCR products were purified enzymatically by digestion with exo-nuclease I and shrimp alkaline phosphatase (Amersham Bio-sciences) and then sequenced using forward and reverse primers according to the original Rhodamine terminator cycle sequencing kit (Applied Biosystems). The full-length of SSI-related genes was obtained by means of RACE. Expression analyses of SLG, SRK and SCR-like genes were performed using mRNA isolated from pistils and anthers by means of quantitative Real-Time PCR with different subdomain-specific primer combinations. Pistils were collected at four developmental stages: T0 (opening flower), and T2, T3 and T4 (2, 3 and 4 days after

Micropropagated olive plants were tested in field to assess their phenotypic behavior. No genetic and morphological differences were detected between micropropagated plants and plants propagated by cuttings. The in vitro protocol that we used for the multiplication of the olive explants insures genetic fidelity and productive potential of the plants.