microRNA159-targeted SlGAMYB transcription factors are required for fruit set in tomato.

The transition from flowering to fruit production, namely fruit set, is crucial to ensure successful sexual plant reproduction. Although studies have described the importance of hormones (i.e. auxin and gibberellins) in controlling fruit set after pollination and fertilization, the role of microRNA-based regulation during ovary development and fruit set is still poorly understood. Here we show that the microRNA159/GAMYB1 and -2 pathway (the miR159/GAMYB1/2 module) is crucial for tomato ovule development and fruit set. MiR159 and SlGAMYBs were expressed in preanthesis ovaries, mainly in meristematic tissues, including developing ovules. SlMIR159-overexpressing tomato cv. Micro-Tom plants exhibited precocious fruit initiation and obligatory parthenocarpy, without modifying fruit shape. Histological analysis showed abnormal ovule development in such plants, which led to the formation of seedless fruits. SlGAMYB1/2 silencing in SlMIR159-overexpressing plants resulted in misregulation of pathways associated with ovule and female gametophyte development and auxin signalling, including AINTEGUMENTA-like genes and the miR167/SlARF8a module. Similarly to SlMIR159-overexpressing plants, SlGAMYB1 was downregulated in ovaries of parthenocarpic mutants with altered responses to gibberellins and auxin. SlGAMYBs likely contribute to fruit initiation by modulating auxin and gibberellin responses, rather than their levels, during ovule and ovary development. Altogether, our results unveil a novel function for the miR159-targeted SlGAMYBs in regulating an agronomically important trait, namely fruit set.

Functional and evolutionary analyses of the miR156 and miR529 families in land plants.

BACKGROUND: MicroRNAs (miRNAs) are important regulatory elements of gene expression. Similarly to coding genes, miRNA genes follow a birth and death pattern of evolution likely reflecting functional relevance and divergence. For instance, miRNA529 is evolutionarily related to miRNA156 (a highly conserved miRNA in land plants), but it is lost in Arabidopsis thaliana. Interestingly, both miRNAs target sequences overlap in some members of the SQUAMOSA promoter-binding protein like (SPL) family, raising important questions regarding the diversification of the miR156/miR529-associated regulatory network in land plants. RESULTS: In this study, through phylogenic reconstruction of miR156/529 target sequences from several taxonomic groups, we have found that specific eudicot SPLs, despite miRNA529 loss, retained the corresponding target site. Detailed molecular evolutionary analyses of miR156/miR529-target sequence showed that loss of miR529 in core eudicots, such as Arabidopsis, is correlated with a more relaxed selection of the miRNA529 specific target element, while miRNA156-specific target sequence is under stronger selection, indicating that these two target sites might be under distinct evolutionary constraints. Importantly, over-expression in Arabidopsis of MIR529 precursor from a monocot, but not from a basal eudicot, demonstrates specific miR529 regulation of AtSPL9 and AtSPL15 genes, which contain conserved responsive elements for both miR156 and miR529. CONCLUSIONS: Our results suggest loss of functionality of MIR529 genes in the evolutionary history of eudicots and show that the miR529-responsive element present in some eudicot SPLs is still functional. Our data support the notion that particular miRNA156 family members might have compensated for the loss of miR529 regulation in eudicot species, which concomitantly may have favored diversification of eudicot SPLs.

Screening and characterization of sex-specific DNA fragments in the freshwater fish matrinchã, Brycon amazonicus (Teleostei: Characiformes: Characidae).

The matrinchã Brycon amazonicus, a commercially important freshwater fish resource, has no heteromorphic sex chromosomes so far described. In the present study, we performed a screening of sex-associated DNA markers in this species, through the use of a random amplified polymorphic DNA (RAPD) assay and a genomic DNA restriction digestion analysis. DNA digestions evidenced no differences between sexes. Sixty-six random primers were used in pooled and individual DNA samples of males and females, and the analysis of the RAPD fingerprints revealed one female sex-associated band. Cloning and sequencing of this band led to the identification of two distinct DNA segments. While one of the isolated fragments showed a significant identity with a described protein gene (phosphatidylinositol glycan anchor biosynthesis, class W), the other fragment, composed of 535 bp, corresponds to a novel DNA marker. Further experiments were performed with this second DNA fragment in order to verify its sex-specificity. Data on dot blot hybridization, using total DNA of both sexes, confirmed its female-specificity in B. amazonicus. A primer set was designed based on its sequence data and used in PCR with DNA samples of this species, leading to diagnose the animals' sexes with a 100 % overall accuracy through a sequence characterized amplified region approach. No amplification results were found for two other species of the genus--B. orbignyanus and B. lundii. The obtained data can lead to the hypothesis that B. amazonicus may present heteromorphic sex chromosomes that should be in an early phase of differentiation.