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Activation of tobacco retrotransposons during tissue culture.

ABSTRACT: Sequences of at least three new families of retrotransposons (Tto1-Tto3) were amplified by PCR from cDNA prepared from protoplasts of an established tobacco cell line, based on the fact that certain amino acids are highly conserved in the reverse transcriptases encoded by retrotransposons. Structural analysis indicates that Tto1 is 5.5 kb long and has features typical of retrotransposons. Transcription of Tto1 starting in the long terminal repeat was active only in cultured cells. Protoplast formation enhanced the transcription. The copy number of Tto1 increased 10-fold in established cell lines; it also increased in plants regenerated from tissue cultures and in transgenic plants. These results indicate that Tto1 is activated during tissue culture. This is the first demonstration of activation of a plant retrotransposon by tissue culture. The copy number of Tto2 and a previously isolated transposon, Tnt1, also increased in established cell lines, indicating that these two retrotransposons may also be activated by tissue culture. These three retrotransposons are cryptic in normally propagated plants: no difference in the copy number was observed between individuals of the same cultivars or even between different cultivars.

SUBMITTER: Hirochika H

PROVIDER: S-EPMC413490 | biostudies-other | 1993 Jun

REPOSITORIES: biostudies-other

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Activation of tobacco retrotransposons during tissue culture.

Hirochika H H

The EMBO journal 19930601 6

Sequences of at least three new families of retrotransposons (Tto1-Tto3) were amplified by PCR from cDNA prepared from protoplasts of an established tobacco cell line, based on the fact that certain amino acids are highly conserved in the reverse transcriptases encoded by retrotransposons. Structural analysis indicates that Tto1 is 5.5 kb long and has features typical of retrotransposons. Transcription of Tto1 starting in the long terminal repeat was active only in cultured cells. Protoplast for ...[more]

PMID: 8389699

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Project description:BackgroundEucalyptus is a highly diverse genus of the Myrtaceae family and widely planted in the world for timber and pulp production. Tissue culture induced callus has become a common tool for Eucalyptus breeding, however, our knowledge about the genes related to the callus maturation and shoot regeneration is still poor.ResultsWe set up an experiment to monitor the callus induction and callus development of two Eucalyptus species - E. camaldulensis (high embryogenic potential) and E. grandis x urophylla (low embryogenic potential). Then, we performed transcriptome sequencing for primary callus, mature callus, shoot regeneration stage callus and senescence callus. We identified 707 upregulated and 694 downregulated genes during the maturation process of the two Eucalyptus species and most of them were involved in the signaling pathways like plant hormone and MAPK. Next, we identified 135 and 142 genes that might play important roles during the callus development of E. camaldulensis and E. grandis x urophylla, respectively. Further, we found 15 DEGs shared by these two Eucalyptus species during the callus development, including Eucgr.D00640 (stem-specific protein TSJT1), Eucgr.B00171 (BTB/POZ and TAZ domain-containing protein 1), Eucgr.C00948 (zinc finger CCCH domain-containing protein 20), Eucgr.K01667 (stomatal closure-related actinbinding protein 3), Eucgr.C00663 (glutaredoxin-C10) and Eucgr.C00419 (UPF0481 protein At3g47200). Interestingly, the expression patterns of these genes displayed "N" shape in the samples. Further, we found 51 genes that were dysregulated during the callus development of E. camaldulensis but without changes in E. grandis x urophylla, such as Eucgr.B02127 (GRF1-interacting factor 1), Eucgr.C00947 (transcription factor MYB36), Eucgr.B02752 (laccase-7), Eucgr.B03985 (transcription factor MYB108), Eucgr.D00536 (GDSL esterase/lipase At5g45920) and Eucgr.B02347 (scarecrow-like protein 34). These 51 genes might be associated with the high propagation ability of Eucalyptus and 22 might be induced after the dedifferentiation. Last, we performed WGCNA to identify the co-expressed genes during the callus development of Eucalyptus and qRT-PCR experiment to validate the gene expression patterns.ConclusionsThis is the first time to globally study the gene profiles during the callus development of Eucalyptus. The results will improve our understanding of gene regulation and molecular mechanisms in the callus maturation and shoot regeneration.

Project description:Tissue culture is an important tool for asexual propagation and genetic transformation of strawberry plants. In plant tissue culture, variation of DNA methylation is a potential source of phenotypic variation in regenerated plants. However, the genome wide dynamic methylation patterns of strawberry tissue culture remain unclear. In this study, we used whole-genome bisulfite sequencing (WGBS) to study genomic DNA methylation changes of a wild strawberry Fragaria nilgerrensis at six stages: from explants of shoot tips to outplanting and acclimation. Global methylation levels showed that CG sites exhibited the highest methylation level in all stages with an average of 49.5%, followed by CHG (33.2%) and CHH (12.4%). Although CHH accounted for the lowest proportion of total cytosine methylation, it showed the most obvious methylation change and the most of these changes occurred in the transposable element regions. The overall methylation levels alternately decreased and increased during the entire tissue culture process and the distribution of DNA methylation was non-uniform among different genetic regions. Furthermore, much more differentially methylated regions (DMRs) were detected in dedifferentiation and redifferentiation stages and most of them were transposable elements, suggesting these processes involved activating or silencing of amounts of transposons. The functional enrichment of the DMR-related genes indicated that genes involved in hormone metabolic processes, plant development and the stress response changed methylation throughout the tissue culture process. Finally, the quantitative real-time PCR (qRT-PCR) was conducted to examine the association of methylation and gene expression of a set of different methylated genes. Our findings give deeper insight into the epigenetic regulation of gene expression during the plant tissue cultures process, which will be useful in the efficient control of somaclonal variations and in crop improvement.

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Activation of tobacco retrotransposons during tissue culture.

Publications

Activation of tobacco retrotransposons during tissue culture.

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