Dataset Information

NEP-TC a rRNA Methyltransferase Involved on Somatic Embryogenesis of Tamarillo (Solanum betaceum Cav.).

ABSTRACT: Somatic embryogenesis (SE) is an important biotechnological tool for large-scale clonal propagation and for embryogenesis research. Moreover, genetic transformation and cryopreservation procedures in many species rely on efficient SE protocols. We have been studying different aspects related to SE induction and somatic embryo development in tamarillo (Solanum betaceum Cav.), a small tree from the Solanaceae family. Previous proteomic analyses identified a protein (NEP-TC, 26.5 kDa) consistently present in non-embryogenic calluses of tamarillo, but absent in the embryogenic ones. In this work, the role of NEP-TC during SE was assessed by gene expression analysis and immunolocalization. The results obtained demonstrated that NEP-TC is a putative member of the SpoU rRNA methylase family. This protein, present in the cytoplasm and nucleus, is expressed in non-embryogenic cells and not expressed in embryogenic cells. Slightly enhanced SE induction levels in tamarillo plants with NEP-TC down-regulated levels also supports the role of this protein on SE induction. Heterologous expression was used to confirm NEP-TC rRNA methyltransferase activity, with enhanced activity levels when rRNA was used as a substrate. These data relate a putative member of the SpoU methylase family with plant morphogenesis, in particular with SE induction.

SUBMITTER: Correia S

PROVIDER: S-EPMC6459958 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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NEP-TC a rRNA Methyltransferase Involved on Somatic Embryogenesis of Tamarillo (<i>Solanum betaceum</i> Cav.).

Correia Sandra S Alhinho Ana T AT Casimiro Bruno B Miguel Célia M CM Oliveira Margarida M Veríssimo Paula P Canhoto Jorge J

Frontiers in plant science 20190405

Somatic embryogenesis (SE) is an important biotechnological tool for large-scale clonal propagation and for embryogenesis research. Moreover, genetic transformation and cryopreservation procedures in many species rely on efficient SE protocols. We have been studying different aspects related to SE induction and somatic embryo development in tamarillo (<i>Solanum betaceum</i> Cav.), a small tree from the Solanaceae family. Previous proteomic analyses identified a protein (NEP-TC, 26.5 kDa) consis ...[more]

PMID: 31024602

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Project description:BackgroundEucalyptus, a highly diverse genus of the Myrtaceae family, is the most widely planted hardwood in the world due to its increasing importance for fiber and energy. Somatic embryogenesis (SE) is one large-scale method to provide commercial use of the vegetative propagation of Eucalyptus and dedifferentiation is a key step for plant cells to become meristematic. However, little is known about the molecular changes during the Eucalyptus SE.ResultsWe compared the transcriptome profiles of the differentiated and dedifferentiated tissues of two Eucalyptus species - E. camaldulensis (high embryogenetic potential) and E. grandis x urophylla (low embryogenetic potential). Initially, we identified 18,777 to 20,240 genes in all samples. Compared to the differentiated tissues, we identified 9229 and 8989 differentially expressed genes (DEGs) in the dedifferentiated tissues of E. camaldulensis and E. grandis x urophylla, respectively, and 2687 up-regulated and 2581 down-regulated genes shared. Next, we identified 2003 up-regulated and 1958 down-regulated genes only in E. camaldulensis, including 6 somatic embryogenesis receptor kinase, 17 ethylene, 12 auxin, 83 ribosomal protein, 28 zinc finger protein, 10 heat shock protein, 9 histone, 122 cell wall related and 98 transcription factor genes. Genes from other families like ABA, arabinogalactan protein and late embryogenesis abundant protein were also found to be specifically dysregulated in the dedifferentiation process of E. camaldulensis. Further, we identified 48,447 variants (SNPs and small indels) specific to E. camaldulensis, including 13,434 exonic variants from 4723 genes (e.g., annexin, GN, ARF and AP2-like ethylene-responsive transcription factor). qRT-PCR was used to confirm the gene expression patterns in both E. camaldulensis and E. grandis x urophylla.ConclusionsThis is the first time to study the somatic embryogenesis of Eucalyptus using transcriptome sequencing. It will improve our understanding of the molecular mechanisms of somatic embryogenesis and dedifferentiation in Eucalyptus. Our results provide a valuable resource for future studies in the field of Eucalyptus and will benefit the Eucalyptus breeding program.

Project description:MicroRNAs (miRNAs) are small noncoding RNAs (18-24 nt) and function in many biological processes in plants. Although Eucalyptus trees are widely planted across the world, our understanding of the miRNA regulation in the somatic embryogenesis (SE) of Eucalyptus is still poor. Here we reported, for the first time, the miRNA profiles of differentiated and dedifferentiated tissues of two Eucalyptus species and identified miRNAs involved in SE of Eucalyptus. Stem and tissue culture-induced callus were obtained from the subculture seedlings of E. camaldulensis and E. grandis x urophylla and were used as differentiated and dedifferentiated samples, respectively. Small RNA sequencing generated 304.2 million clean reads for the Eucalyptus samples (n = 3) and identified 888 miRNA precursors (197 known and 691 novel) for Eucalyptus. These miRNAs were mainly distributed in chromosomes Chr03, Chr05, and Chr08 and can produce 46 miRNA clusters. Then, we identified 327 and 343 differentially expressed miRNAs (DEmiRs) in the dedifferentiation process of E. camaldulensis and E. grandis x urophylla, respectively. DEmiRs shared by the two Eucalyptus species might be involved in the development of embryonic callus, such as MIR156, MIR159, MIR160, MIR164, MIR166, MIR169, MIR171, MIR399, and MIR482. Notably, we identified 81 upregulated and 67 downregulated miRNAs specific to E. camaldulensis, which might be associated with the high embryogenic potential. Target prediction and functional analysis showed that they might be involved in longevity regulating and plant hormone signal transduction pathways. Further, using the gene expression profiles, we observed the negative regulation of miRNA-target pairs, such as MIR160~ARF18, MIR396~GRF6, MIR166~ATHB15/HD-ZIP, and MIR156/MIR157~SPL1. Interestingly, transcription factors such as WRKY, MYB, GAMYB, TCP4, and PIL1 were found to be regulated by the DEmiRs. The genes encoding PIL1 and RPS21C, regulated by upregulated miRNAs (e.g., egd-N-miR63-5p, egd-N-miR63-5p, and MIR169,) were downregulated exclusively in the dedifferentiation of E. camaldulensis. This is the first time to study the miRNA regulation in the dedifferentiation process of Eucalyptus and it will provide a valuable resource for future studies. More importantly, it will improve our understanding of miRNA regulation during the somatic embryogenesis of Eucalyptus and benefit the Eucalyptus breeding program.

Project description:BACKGROUND: In our laboratory we use cultured chicory (Cichorium intybus) explants as a model to investigate cell reactivation and somatic embryogenesis and have produced 2 chicory genotypes (K59, C15) sharing a similar genetic background. K59 is a responsive genotype (embryogenic) capable of undergoing complete cell reactivation i.e. cell de- and re-differentiation leading to somatic embryogenesis (SE), whereas C15 is a non-responsive genotype (non-embryogenic) and is unable to undergo SE. Previous studies 1 showed that the use of the beta-D-glucosyl Yariv reagent (beta-GlcY) that specifically binds arabinogalactan-proteins (AGPs) blocked somatic embryo production in chicory root explants. This observation indicates that beta-GlcY is a useful tool for investigating somatic embryogenesis (SE) in chicory. In addition, a putative AGP (DT212818) encoding gene was previously found to be significantly up-regulated in the embryogenic K59 chicory genotype as compared to the non-embryogenic C15 genotype suggesting that this AGP could be involved in chicory re-differentiation 2. In order to improve our understanding of the molecular and cellular regulation underlying SE in chicory, we undertook a detailed cytological study of cell reactivation events in K59 and C15 genotypes, and used microarray profiling to compare gene expression in these 2 genotypes. In addition we also used beta-GlcY to block SE in order to identify genes potentially involved in this process. RESULTS: Microscopy confirmed that only the K59, but not the C15 genotype underwent complete cell reactivation leading to SE formation. beta-GlcY-treatment of explants blocked in vitro SE induction, but not cell reactivation, and induced cell wall modifications. Microarray analyses revealed that 78 genes were differentially expressed between induced K59 and C15 genotypes. The expression profiles of 19 genes were modified by beta-GlcY-treatment. Eight genes were both differentially expressed between K59 and C15 genotypes during SE induction and transcriptionally affected by beta-GlcY-treatment: AGP (DT212818), 26 S proteasome AAA ATPase subunit 6 (RPT6), remorin (REM), metallothionein-1 (MT1), two non-specific lipid transfer proteins genes (SDI-9 and DEA1), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), and snakin 2 (SN2). These results suggest that the 8 genes, including the previously-identified AGP gene (DT212818), could be involved in cell fate determination events leading to SE commitment in chicory. CONCLUSION: The use of two different chicory genotypes differing in their responsiveness to SE induction, together with beta-GlcY-treatment represented an efficient tool to discriminate cell reactivation from the SE morphogenetic pathway. Such an approach, together with microarray analyses, permitted us to identify several putative key genes related to the SE morphogenetic pathway in chicory.

Dataset Information

NEP-TC a rRNA Methyltransferase Involved on Somatic Embryogenesis of Tamarillo (Solanum betaceum Cav.).

Publications

NEP-TC a rRNA Methyltransferase Involved on Somatic Embryogenesis of Tamarillo (<i>Solanum betaceum</i> Cav.).

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