Genome-wide analysis of AGO, DCL and RDR gene families reveals RNA-directed DNA methylation is involved in fruit abscission in Citrus sinensis

Background: Small RNAs regulate a wide variety of processes in plants, from organ development to both biotic and abiotic stress response. Being master regulators in genetic networks, their biogenesis and action is a fundamental aspect to characterize in order to understand plant growth and development. Three main gene families are critical components of RNA silencing: DICER-LIKE (DCL), ARGONAUTE (AGO) and RNA-DEPENDENT RNA POLYMERASE (RDR). Even though they have been characterized in other plant species, there is no information about these gene families in Citrus sinensis, one of the most important fruit species from both economical and nutritional reasons. While small RNAs have been implicated in the regulation of multiple aspects of plant growth and development, their role in the abscission process has not been characterized yet. Results: Using genome-wide analysis and a phylogenetic approach, we identified a total of 13 AGO, 5 DCL and 7 RDR genes. We characterized their expression patterns in root, leaf, flesh, peel and embryo samples using RNA-seq data. Moreover, we studied their role in fruit abscission through gene expression analysis in fruit rind compared to abscission zone from samples obtained by laser capture microdissection. Interestingly, we determined that the expression of several RNA silencing factors are down-regulated in fruit abscission zone, being particularly represented gene components of the RNA-dependent DNA Methylation pathway, indicating that repression of this process is necessary for fruit abscission to take place in Citrus sinensis. Conclusions: The members of these 3 families present characteristic conserved domains and distinct expression patterns. We provide a detailed analysis of the members of these families and improved the annotation of some of these genes based on RNA-seq data. Our data suggests that the RNA-dependent DNA Methylation pathway is involved in the important fruit abscission process in C. sinensis.Fil: Sabbione, Agustín Andrés. Universidad Nacional del Litoral. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Daurelio, Lucas Damian. Universidad Nacional del Litoral. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Veggeti, A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Ciencias Agrarias; ArgentinaFil: Talón, Manuel. Institut Valencià Dinvestigacions Agràries; EspañaFil: Tadeo, Francisco. Institut Valencià Dinvestigacions Agràries; EspañaFil: Dotto, Marcela Claudia. Universidad Nacional del Litoral. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentin

Developmental reprogramming by UV-B radiation in plants

Plants are extremely plastic organisms with the ability to adapt and respond to the changing environmental conditions surrounding them. Sunlight is one of the main resources for plants, both as a primary energy source for photosynthesis and as a stimulus that regulates different aspects of their growth and development. UV-B comprises wavelengths that correspond to a high energy region of the solar spectrum capable of reaching the biosphere, influencing plant growth. It is currently believed that plants are able to acclimate when growing under the influence of this radiation and perceive it as a signal, without stress signs. Nonetheless, many UV-B induced changes are elicited after DNA damage occurs as a consequence of exposure. In this review we focus on the influence of UV-B on leaf, flower and root development and emphasize the limited understanding of the molecular mechanisms for most of this developmental processes affected by UV-B documented over the years of research in this area.Fil: Dotto, Marcela Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Ciencias Agrarias; ArgentinaFil: Casati, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentin

UV-B radiation delays flowering time through changes in the PRC2 complex activity and miR156 levels in Arabidopsis thaliana

UV-B is a high-energy component of the solar radiation perceived by the plant and induces a number of modifications in plant growth and development, including changes in flowering time. However, the molecular mechanisms underlying these changes are largely unknown. In the present work, we demonstrate that Arabidopsis plants grown under white light supplemented with UV-B show a delay in flowering time, and this developmental reprogramming is mediated by the UVR8 photoreceptor. Using a combination of gene expression analyses and UV-B irradiation of different flowering mutants, we gained insight into the pathways involved in the observed flowering time delay in UV-B-exposed Arabidopsis plants. We provide evidence that UV-B light downregulates the expression of MSI1 and CLF, two of the components of the polycomb repressive complex 2, which in consequence drives a decrease in H3K27me3 histone methylation of MIR156 and FLC genes. Modification in the expression of several flowering time genes as a consequence of the decrease in the polycomb repressive complex 2 activity was also determined. UV-B exposure of flowering mutants supports the involvement of this complex in the observed delay in flowering time, mostly through the age pathway.Fil: Dotto, Marcela Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; ArgentinaFil: Gomez, Maria Sol. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; ArgentinaFil: Soto, María Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; ArgentinaFil: Casati, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentin

An experimental protocol for teaching CRISPR/Cas9 in a post-graduate plant laboratory course: An analysis of mutant-edited plants without sequencing

The CRISPR/Cas9 system is widely used for editing genes in various organisms and is a very useful tool due to its versatility, simplicity, and efficiency. To teach its principles to post-graduate students we designed a laboratory activity to obtain and analyze PDS3 mutants in Arabidopsis thaliana plants consisting of: 1) Design of guide RNAs using bioinformatics tools; 2) plant transformation (which is optional depending on the length of the course); 3) observation and evaluation of the mutant's phenotypes in the Phytoene desaturase (PDS3) gene, which exhibit an albino phenotype and different degrees of mosaicism in the editing events we evaluated; 4) PCR amplification of a fragment that includes the mutated region followed by analysis of single-stranded DNA conformation polymorphisms (SSCP) using native polyacrylamide gel electrophoresis and silver nitrate staining to detect changes in the amplicon sequence due to gene editing. Through SSCP, the students were able to distinguish between homozygous and heterozygous edited plants. A highlight feature of this protocol is the visualization and detection of the mutation/edition without sequencing the edited fragment.Fil: Mayta, Martín Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas.; Argentina. Universidad Adventista del Plata. Facultad de Ciencias de la Salud; ArgentinaFil: Dotto, Marcela Claudia. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Instituto de Ciencias Agropecuarias del Litoral. - Universidad Nacional del Litoral. Instituto de Ciencias Agropecuarias del Litoral.; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas.; ArgentinaFil: Krapp, Adriana del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas.; ArgentinaFil: Orellano, Elena Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas.; Argentin

Influence of plant growth regulators on Expansin2 expression in strawberry fruit. Cloning and functional analysis of FaEXP2 promoter region

FaEXP2 encodes a fruit-specific expansin that is known to be associated with cell wall loosening and fruit softening in strawberry (Fragaria × ananassa Duch.). In spite of its relevant role in fruit ripening, little is known about the hormonal regulation of this gene. In the present study, we isolated a 650 bp fragment of the FaEXP2 gene promoter and the in silico analysis revealed the presence of cis-acting elements associated with hormones, light and stress-related responses. With the aim of characterizing the hormonal regulation of FaEXP2 expression, strawberry fruit were treated with plant growth regulators and changes in transcript levels were analyzed by qPCR. FaEXP2 expression levels were significantly higher than control in fruit treated with abscisic acid (ABA), and when the endogenous source of auxins (achenes) was removed. In contrast, transcript accumulation was not affected by exogenous applications of gibberellic acid (GA3), naphtalenacetic acid (NAA), ethylene or 1-methylcyclopropene (1-MCP). Also, functional analysis of FaEXP2 gene promoter was performed by transient and permanent strawberry transformation. Histochemical GUS staining analysis showed that the 650 bp-promoter fragment was able to drive the reporter gene expression specifically to the receptacle and along ripening. Our results provide new insights into the hormonal regulation of FaEXP2 expression.Fil: Nardi, Cristina Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Villarreal, Natalia Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Dotto, Marcela Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Ariza, M. T.. Universidad de Málaga; EspañaFil: Vallarino, J. G.. Universidad de Málaga; EspañaFil: Martinez, Gustavo Adolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; ArgentinaFil: Valpuesta, V.. Universidad de Málaga; EspañaFil: Civello, Pedro Marcos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; Argentin