Comparative transcriptome analysis of stylar canal cells identifies novel candidate genes implicated in the self-incompatibility response of Citrus clementina

Marco Caruso Paz Merelo Gaetano Distefano Stefano La Malfa Angela Roberta Lo Piero Francisco R Tadeo Manuel Talon Alessandra Gentile Background: Reproductive biology in citrus is still poorly understood. Although in recent years several efforts have been made to study pollen-pistil interaction and self-incompatibility, little information is available about the molecular mechanisms regulating these processes. Here we report the identification of candidate genes involved in pollen-pistil interaction and self-incompatibility in clementine (Citrus clementina Hort. ex Tan.). These genes have been identified comparing the transcriptomes of laser-microdissected stylar canal cells (SCC) isolated from two genotypes differing for self-incompatibility response ('Comune', a self-incompatible cultivar and 'Monreal', a selfcompatible mutation of 'Comune'). Results: The transcriptome profiling of SCC indicated that the differential regulation of few specific, mostly uncharacterized transcripts is associated with the breakdown of self-incompatibility in 'Monreal'. Among them, a novel F-box gene showed a drastic up-regulation both in laser microdissected stylar canal cells and in selfpollinated whole styles with stigmas of 'Comune' in concomitance with the arrest of pollen tube growth. Moreover, we identify a non-characterized gene family as closely associated to the self-incompatibility genetic program activated in 'Comune'. Three different aspartic-acid rich (Asp-rich) protein genes, located in tandem in the clementine genome, were over-represented in the transcriptome of 'Comune'. These genes are tightly linked to a DELLA gene, previously found to be up-regulated in the self-incompatible genotype during pollen-pistil interaction. Conclusion: The highly specific transcriptome survey of the stylar canal cells identified novel genes which have not been previously associated with self-pollen rejection in citrus and in other plant species. Bioinformatic and transcriptional analyses suggested that the mutation leading to self-compatibility in 'Monreal' affected the expression of non-homologous genes located in a restricted genome region. Also, we hypothesize that the Asprich protein genes may act as Ca2+ entrapping proteins, potentially regulating Ca2+ homeostasis during self-pollen recognition. - Background Among the citrus species, several pummelos (Citrus grandis L. Osbeck) and mandarin-like varieties are selfincompatible [1]. Clementine mandarin (Citrus clementina Hort. ex Tan.), derived from an uncontrolled cross between a sweet orange and a mandarin, is probably the * Correspondence: 1Dipartimento di Scienze delle Produzioni Agrarie e Alimentari, Universit degli Studi di Catania, Via Valdisavoia 5, 95123 Catania, Italy Full list of author information is available at the end of the article most widespread citrus species showing self-incompatibility (SI). It is characterized by gametophytic SI, with the pollen tubes stopping their growth in the upper or middle style [2,3]. Moreover, in this species SI is coupled with a variable degree of parthenocarpy. SI and parthenocarpy in citrus are very important traits for fruit production because they result in seedless fruits, which have a higher value in the markets compared to the seeded ones. Therefore, understanding the molecular basis of SI would be important to plan marker-assisted breeding to obtain new seedless genotypes. Despite the importance of this trait, the genetic basis are still poorly understood and the key genes of SI have not been identified yet. The study of populations segregating for SI might be definitely a powerful strategy to give new insights into its genetic basis. However, the difficulties to obtain and characterize appropriate populations, with a variable degree of parthenocarpy and female/male sterility observed in the progeny might limit this approach. Such kind of strategy for the identification of the S-locus was carried out analyzing several crosses among different citrus cultivar and accessions with Got-3 isozyme, which is thought to be linked with the S-locus [4], providing only a rough estimation of their possible S-genotype. In recent times, different research groups attempted to better understand SI and pollen-pistil interaction in several citrus genotypes, mainly trying to characterize putative homologs of key genes and proteins of already characterized SI systems. Gentile and colleagues [5] reported the involvement of Ca2+-dependent transglutaminase (TGase) in the self-incompatible response in pummelo, as already reported for Rosaceae [6]. Regarding the S-locus genes, a S-like RNase has been isolated from Zigui shatian pummelo [7], however the authors suggested that this gene might play an important role during ovary senescence rather than in the incompatibility response. Another S-like RNase has been isolated from a mandarin variety and was partially characterized [8], but its not still clear whether this gene is the key determinant for the self-incompatible response. To overcome these limits, the transcriptome analysis of natural mutants displaying contrasting compatibility behaviour might be more effective to better understanding the molecular basis controlling the progamic phase in citrus. Over the last decade, the genome and/or transcriptome analysis of natural or induced citrus mutants have been a powerful strategy to study the molecular basis of agronomically important traits, such as ripening period, fruit pigmentation, seedlessness and other traits related to quality [9-12]. Concerning SI, a few citrus natural mutants displaying different sexual behaviour with respect to their original varieties have been identified and characterized [13-16]. Differences between the mutants and the original cultivars were related to differences in pollen [13], style [15] or ovary [14] functionality. In some cases, different behaviours during the progamic phase were associated to abnormal embryo development [13,16]. Therefore, it seems clear the mechanisms preventing fertilization are different in these genotypes, so its reasonable to hypothesize that the mutations affected different genes or pathways implicated in reproduction. Recently we chose two clementine clones with contrasting behaviour relating to self-pollen recognition (Comune, self-incompatible; and Monreal, self-compatible mutation of Comune [17]) as a model to identify candidate genes implicated in pollen-pistil interaction. Histological assays and analysis of pollen tube kinetics were performed to study the pollen tube behaviour in the two genotypes, and to assess whether the breakdown of SI in Monreal was caused by changes in pistil or pollen functionality. The analysis demonstrated that the Monreal mutation affected pistil functions, since pollen tubes of the two varieties grew equally in the pistils of self-compatible mutant, while Comune rejected the pollen of both varieties, recognizing the pollen of Monreal as self-pollen [15]. A f (...truncated)