No abstract

Small non-coding RNAs (sncRNAs) are molecules with important regulatory functions during development and environmental responses across all groups of terrestrial plants. In seed plants, the development of a mature embryo from the zygote follows a synchronized cell division sequence, and growth and differentiation events regulated by highly regulated gene expression. However, given the distinct features of the initial stages of embryogenesis in gymnosperms and angiosperms, it is relevant to investigate to what extent such differences emerge from differential regulation mediated by sncRNAs. Within these, the microRNAs (miRNAs) are the best characterized class, and while many miRNAs are conserved and significantly represented across angiosperms and other seed plants during embryogenesis, some miRNA families are specific to some plant lineages. Being a model to study zygotic embryogenesis and a relevant biotechnological tool, we systematized the current knowledge on the presence and characterization of miRNAs in somatic embryogenesis (SE) of seed plants, pinpointing the miRNAs that have been reported to be associated with SE in angiosperm and gymnosperm species. We start by conducting an overview of sncRNA expression profiles in the embryonic tissues of seed plants. We then highlight the miRNAs described as being involved in the different stages of the SE process, from its induction to the full maturation of the somatic embryos, adding references to zygotic embryogenesis when relevant, as a contribution towards a better understanding of miRNA-mediated regulation of SE.

Plant regeneration is a well-known capacity of plants occurring either in vivo or in vitro. This potential is the basis for plant micropropagation and genetic transformation as well as a useful system to analyse different aspects of plant development. Recent studies have proven that RNA species with no protein-coding capacity are key regulators of cellular function and essential for cell reprogramming. In this review, the current knowledge on the role of several ncRNAs in plant regeneration processes is summarized, with a focus on cell fate reprogramming. Moreover, the involvement/impact of microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and small-interfering RNAs (siRNAs) in the regulatory networks of cell dedifferentiation, proliferation and differentiation is also analysed. A deeper understanding of plant ncRNAs in somatic cell reprogramming will allow a better modulation of in vitro regeneration processes such as organogenesis and somatic embryogenesis.

No abstract

Somatic embryogenesis in Solanum betaceum (tamarillo) has proven to be an effective model system for studying morphogenesis, since optimized plant regeneration protocols are available, and embryogenic competent cell lines can be induced from different explants. Nevertheless, an efficient genetic transformation system for embryogenic callus (EC) has not yet been implemented for this species. Here, an optimized faster protocol of genetic transformation using Agrobacterium tumefaciens is described for EC. The sensitivity of EC to three antibiotics was determined, and kanamycin proved to be the best selective agent for tamarillo callus. Two Agrobacterium strains, EHA105 and LBA4404, both harboring the p35SGUSINT plasmid, carrying the reporter gene for β-glucuronidase (gus) and the marker gene neomycin phosphotransferase (nptII), were used to test the efficiency of the process. To increase the success of the genetic transformation, a cold-shock treatment, coconut water, polyvinylpyrrolidone and an appropriate selection schedule based on antibiotic resistance were employed. The genetic transformation was evaluated by GUS assay and PCR-based techniques, and a 100% efficiency rate was confirmed in the kanamycin-resistant EC clumps. Genetic transformation with the EHA105 strain resulted in higher values for gus insertion in the genome. The protocol presented provides a useful tool for functional gene analysis and biotechnology approaches.

No abstract

O avanço nas tecnologias de sequenciação e caracterização do RNA tem permitido revelar mecanismos essenciais na biologia dos organismos até recentemente desconhecidos.O transcritoma das células engloba uma grande diversidade de formas de RNA, que se agrupam em RNA codificante e não codificante. O primeiro é o RNA mensageiro (mRNA) que tem como função servir de modelo para a síntese de proteínas na célula. Por sua vez, o RNA não codificante, constitui 98% do transcritoma e inclui não só o RNA de transferência (tRNA) e o RNA ribossomal (rRNA), essenciais na tradução, mas também os RNAs nucleolares (snoRNAs), Vault RNAs (vRNAs), PIWI-interacting RNAs (piRNAs) e ainda os pequenos RNAs (sRNAs), onde se incluem várias formas de RNAs de interferência (siRNAs) e microRNAs (miRNAs).Os microRNAs são das mais pequenas moléculas de RNA não codificante, sendo constituídos apenas por 20-24 nucleótidos. Foram descobertos em 1993 em estudos no nemátode Caenorhabditis elegans, aquando da análise dos genes lin-14 e lin-4, envolvidos no desenvolvimento deste organismo modelo 1 . Nas plantas, o primeiro miRNA foi identificado em Arabidopsis thaliana (L.) Heynh. 2 e até à data foram já identificadas mais de cerca de 8500 destas moléculas em mais de 70 espécies. A expressão dos miRNAs é altamente dependente do tipo de tecido e, por isso, os seus padrões e timings de expressão variam ao longo do desenvolvimento. Estão envolvidos no controlo da expressão genética, atuando pós--transcricionalmente. Por exemplo, em mamíferos, a expressão de 60% dos genes codificantes de proteínas é regulada por miRNAs 3 . Muitos miRNAs pertencem a famílias cujas sequências são muito similares entre os membros, variando apenas num único nucleótido.Por sua vez, cada miRNA atua em vários mRNAs-alvo, sendo que nas plantas os miRNAs têm, em geral, menos genes-alvo que os animais. Além disso, cada mRNA é alvo da ação de diferentes miRNAs, resultando assim numa complexa rede de regulação 4,5 . Os miRNAs são codificados pelos genes MIRNAs (MIR), muitos dos quais bastante conservados no Reino Vegetal 6 . Transcritos pela RNA POLIMERASE II, os transcritos MIR de cadeia simples, dobram-se sobre si mesmos emparelhando e formando uma estrutura semelhante a um gancho de cabelo (harpin ou stem-loop), passando assim a designar-se miRNAs primários (pri-miRNAs) 7 . Nas plantas, os pri-miRNAs são clivados pela ação da RNase DICER-LIKE 1 (DCL1), daí resultando pequenas porções de RNA de cadeia dupla, os precursores dos miRNAs (pre-miRNAs). Estes sofrem depois metilação nas extremidades 3' pela ação da RNA metiltransferase HUA ENHANCER 1 (HEN1), tornando-se maduros e, consequentemente, saem do núcleo através de uma proteína homóloga da exportina, designada HASTY 8 . Uma vez no citoplasma, ligam-se à proteína ARGONAUTE 1 (AGO1) e, em conjunto com outras proteínas, formam o complexo de silenciamento induzido por RNA CITAÇÃO

Long-read sequencing methods allow a comprehensive analysis of transcriptomes in identifying full-length transcripts. This revolutionary method represents a considerable breakthrough for non-model species since it allows enhanced gene annotation and gene expression studies when compared to former sequencing methods. However, woody plant tissues are challenging to the successful preparation of cDNA libraries, thus, impairing further cutting-edge sequencing analyses. Here, a detailed protocol for preparing cDNA libraries suitable for high throughput RNA sequencing using Oxford Nanopore Technologies® is described. This method was used to prepare eight barcoded cDNA libraries from two Solanum betaceum cell lines: one with compact morphology and embryogenic competency (EC) and another with friable and non-embryogenic (NEC). The libraries were successfully sequenced, and data quality assessment showed high mean quality scores. Using this method, long-read sequencing will allow a comprehensive analysis of plant transcriptomes.