Exploring N6-methyladenosine (m6A) modification in tree species: opportunities and challenges.

N 6-methyladenosine (m6A) in eukaryotes is the most common and widespread internal modification in mRNA. The modification regulates mRNA stability, translation efficiency, and splicing, thereby fine-tuning gene regulation. In plants, m6A is dynamic and critical for various growth stages, embryonic development, morphogenesis, flowering, stress response, crop yield, and biomass. Although recent high-throughput sequencing approaches have enabled the rapid identification of m6A modification sites, the site-specific mechanism of this modification remains unclear in trees. In this review, we discuss the functional significance of m6A in trees under different stress conditions and discuss recent advancements in the quantification of m6A. Quantitative and functional insights into the dynamic aspect of m6A modification could assist researchers in engineering tree crops for better productivity and resistance to various stress conditions.

Low phosphorus impact on Moso bamboo (Phyllostachys edulis) root morphological polymorphism and expression pattern of the related genes.

Moso bamboo typically grows in phosphorus (P)-deficient soil that limits its growth and development. In this study, 10 Moso bamboo genotypes (Ph-1 to Ph-10) were evaluated for their responses to P deficiency during the seedling stage by growing them in both P-sufficient and P-deficient conditions. Adaptive responses to low P (LP) conditions were observed in the majority of genotypes. Under P deficiency conditions, the total biomass decreased in several genotypes, but at the same time, the root-to-shoot ratio increased. Principal component analysis identified two main comprehensive traits (PC1 and PC2) related to the root volume and surface area and P concentration and accumulation. Based on the analysis, two genotypes (Ph-6 and Ph-10) were identified with significantly different levels of tolerance to P deficiency. The results revealed that the genotype Ph-10 responded to P deficiency by significantly increasing the root surface area and volume, while simultaneously reducing the number of root cortex cells when compared with the genotype Ph-6, which showed the lowest tolerance (intolerant). The genotype Ph-10 exhibited a robust response to external LP conditions, marked by elevated expression levels of PHOSPHATE TRANSPORTERs and SYG1/PHO81/XPR1s. In situ Polymerase Chain Reaction (PCR) analysis also revealed distinct tissue-specific expression patterns of the genes in the roots, particularly highlighting the differences between Ph-6 and Ph-10. The results provide a foundation for elucidating the mechanism of LP tolerance, thus potentially contributing to developing high P-use efficiency in Moso bamboo species.

Epigenetic modifications and miRNAs determine the transition of somatic cells into somatic embryos.

KEY MESSAGE: This review discusses the epigenetic changes during somatic embryo (SE) development, highlights the genes and miRNAs involved in the transition of somatic cells into SEs as a result of epigenetic changes, and draws insights on biotechnological opportunities to study SE development. Somatic embryogenesis from somatic cells occurs in a series of steps. The transition of somatic cells into somatic embryos (SEs) is the most critical step under genetic and epigenetic regulations. Major regulatory genes such as SERK, WUS, BBM, FUS3/FUSA3, AGL15, and PKL, control SE steps and development by turning on and off other regulatory genes. Gene transcription profiles of somatic cells during SE development is the result of epigenetic changes, such as DNA and histone protein modifications, that control and decide the fate of SE formation. Depending on the type of somatic cells and the treatment with plant growth regulators, epigenetic changes take place dynamically. Either hypermethylation or hypomethylation of SE-related genes promotes the transition of somatic cells. For example, the reduced levels of DNA methylation of SERK and WUS promotes SE initiation. Histone modifications also promote SE induction by regulating SE-related genes in somatic cells. In addition, miRNAs contribute to the various stages of SE by regulating the expression of auxin signaling pathway genes (TIR1, AFB2, ARF6, and ARF8), transcription factors (CUC1 and CUC2), and growth-regulating factors (GRFs) involved in SE formation. These epigenetic and miRNA functions are unique and have the potential to regenerate bipolar structures from somatic cells when a pluripotent state is induced. However, an integrated overview of the key regulators involved in SE development and downstream processes is lacking. Therefore, this review discusses epigenetic modifications involved in SE development, SE-related genes and miRNAs associated with epigenetics, and common cis-regulatory elements in the promoters of SE-related genes. Finally, we highlight future biotechnological opportunities to alter epigenetic pathways using the genome editing tool and to study the transition mechanism of somatic cells.

Strigolactone Mediates Moso Bamboo Root Response to Phosphate Stress.

Both phosphate (Pi) deficiency and high Pi significantly affect moso bamboo growth and degrade bamboo forests. A novel plant hormone, strigolactone (SL), plays a crucial role in root growth under low Pi, but the SL regulatory mechanism has not been systematically reported in moso bamboo. In our study, we investigated the SL-mediated root growth mechanism in response to Pi stress in moso bamboo. With the decrease of the Pi level, 5-deoxystrigol and strigol significantly increased in the root exudates. Transcriptome sequencing of the primary root tip and lateral root primordium zone (LRP) under low, sufficient, and high Pi indicated that SL-biosynthesis and -signaling changes are part of the early root responses. The effects of the SL analogue (rac-GR24) and SL inhibitor (TIS108) on the root architecture under low and high Pi revealed that SL mediates bamboo root responses by regulating its biosynthesis and signal transduction and influencing other hormone pathways.

A long non-coding RNA PelncRNA1 is involved in Phyllostachys edulis response to UV-B stress.

Phyllostachys edulis (moso bamboo) is China's most widespread bamboo species, with significant economic and ecological values. Long non-coding RNA (lncRNA) is a type of regulatory RNA that is longer than 200 nucleotides and incapable of encoding proteins, and is frequently involved in regulating biotic and abiotic stress and plant development. However, the biological functions of lncRNA in moso bamboo are unknown. In this study, a lncRNA (named PelncRNA1) differentially expressed following UV-B treatment was discovered in the whole transcriptome sequencing database of moso bamboo. The target genes were filtered and defined by correlation analysis of PelncRNA1 and gene expression pattern. The expression levels of PelncRNA1 and its target genes were verified using qRT-PCR. The results demonstrated that the expression levels of PelncRNA1 and its target genes increased during UV-B treatment. In Arabidopsis transgenic seedlings and moso bamboo protoplasts, PelncRNA1 was discovered to influence the expression of its target genes when overexpressed. In addition, transgenic Arabidopsis showed higher tolerance to UV-B stress. These results suggest that PelncRNA1 and its target genes are involved in the response of moso bamboo to UV-B stress. The novel findings would contribute to our understanding of how lncRNAs regulate the response to abiotic stresses in moso bamboo.

Effects of different phosphorus levels on tiller bud development in hydroponic Phyllostachys edulis seedlings.

An appropriate amount of phosphate fertilizer can improve the germination rate of bamboo buds and increase the bamboo shoot output. However, the underlying biological mechanisms of phosphate fertilizer in bamboo shoot development have not been systematically reported. Herein, the effects of low (LP, 1 µM), normal (NP, 50 µM) and high (HP, 1000 µM) phosphorus (P) on the growth and development of moso bamboo (Phyllostachys edulis) tiller buds were first investigated. Phenotypically, the seedling biomass, average number of tiller buds and bud height growth rate under the LP and HP treatments were significantly lower than those under the NP treatment. Next, the microstructure difference of tiller buds in the late development stage (S4) at three P levels was analyzed. The number of internode cells and vascular bundles were significantly lower in the LP treatments than in the NP treatments. The relative expression levels of eight P transport genes, eight hormone-related genes and four bud development genes at the tiller bud developmental stage (S2-S4) and the tiller bud re-tillering stage were analyzed with real-time polymerase chain reaction. The results showed that the expression trends for most P transport genes, hormone-related genes and bud development genes from S2 to S4 were diversified at different P levels, and the expression levels were also different at different P levels. In the tiller bud re-tillering stage, the expression levels of seven P transport genes and six hormone-related genes showed a downward trend with increasing P level. REV expression level decreased under LP and HP conditions. TB1 expression level increased under HP condition. Therefore, we conclude that P deficiency inhibits tiller bud development and re-tillering, and that P depends on the expression of REV and TB1 genes and auxin, cytokinin and strigolactones synthesis and transporter genes to mediate tiller bud development and re-tillering.

The role of LTR retrotransposons in plant genetic engineering: how to control their transposition in the genome.

KEY MESSAGE: We briefly discuss that the similarity of LTR retrotransposons to retroviruses is a great opportunity for the development of a genetic engineering tool that exploits intragenic elements in the plant genome for plant genetic improvement. Long terminal repeat (LTR) retrotransposons are very similar to retroviruses but do not have the property of being infectious. While spreading between its host cells, a retrovirus inserts a DNA copy of its genome into the cells. The ability of retroviruses to cause infection with genome integration allows genes to be delivered to cells and tissues. Retrovirus vectors are, however, only specific to animals and insects, and, thus, are not relevant to plant genetic engineering. However, the similarity of LTR retrotransposons to retroviruses is an opportunity to explore the former as a tool for genetic engineering. Although recent long-read sequencing technologies have advanced the knowledge about transposable elements (TEs), the integration of TEs is still unable either to control them or to direct them to specific genomic locations. The use of existing intragenic elements to achieve the desired genome composition is better than using artificial constructs like vectors, but it is not yet clear how to control the process. Moreover, most LTR retrotransposons are inactive and unable to produce complete proteins. They are also highly mutable. In addition, it is impossible to find a full active copy of a LTR retrotransposon out of thousands of its own copies. Theoretically, if these elements were directly controlled and turned on or off using certain epigenetic mechanisms (inducing by stress or infection), LTR retrotransposons could be a great opportunity to develop a genetic engineering tool using intragenic elements in the plant genome. In this review, the recent developments in uncovering the nature of LTR retrotransposons and the possibility of using these intragenic elements as a tool for plant genetic engineering are briefly discussed.

Epigenetic stress memory: A new approach to study cold and heat stress responses in plants.

Understanding plant stress memory under extreme temperatures such as cold and heat could contribute to plant development. Plants employ different types of stress memories, such as somatic, intergenerational and transgenerational, regulated by epigenetic changes such as DNA and histone modifications and microRNAs (miRNA), playing a key role in gene regulation from early development to maturity. In most cases, cold and heat stresses result in short-term epigenetic modifications that can return to baseline modification levels after stress cessation. Nevertheless, some of the modifications may be stable and passed on as stress memory, potentially allowing them to be inherited across generations, whereas some of the modifications are reactivated during sexual reproduction or embryogenesis. Several stress-related genes are involved in stress memory inheritance by turning on and off transcription profiles and epigenetic changes. Vernalization is the best example of somatic stress memory. Changes in the chromatin structure of the Flowering Locus C (FLC) gene, a MADS-box transcription factor (TF), maintain cold stress memory during mitosis. FLC expression suppresses flowering at high levels during winter; and during vernalization, B3 TFs, cold memory cis-acting element and polycomb repressive complex 1 and 2 (PRC1 and 2) silence FLC activation. In contrast, the repression of SQUAMOSA promoter-binding protein-like (SPL) TF and the activation of Heat Shock TF (HSFA2) are required for heat stress memory. However, it is still unclear how stress memory is inherited by offspring, and the integrated view of the regulatory mechanisms of stress memory and mitotic and meiotic heritable changes in plants is still scarce. Thus, in this review, we focus on the epigenetic regulation of stress memory and discuss the application of new technologies in developing epigenetic modifications to improve stress memory.

Retrotransposons: How the continuous evolutionary front shapes plant genomes for response to heat stress.

Long terminal repeat retrotransposons (LTR retrotransposons) are the most abundant group of mobile genetic elements in eukaryotic genomes and are essential in organizing genomic architecture and phenotypic variations. The diverse families of retrotransposons are related to retroviruses. As retrotransposable elements are dispersed and ubiquitous, their "copy-out and paste-in" life cycle of replicative transposition leads to new genome insertions without the excision of the original element. The overall structure of retrotransposons and the domains responsible for the various phases of their replication is highly conserved in all eukaryotes. The two major superfamilies of LTR retrotransposons, Ty1/Copia and Ty3/Gypsy, are distinguished and dispersed across the chromosomes of higher plants. Members of these superfamilies can increase in copy number and are often activated by various biotic and abiotic stresses due to retrotransposition bursts. LTR retrotransposons are important drivers of species diversity and exhibit great variety in structure, size, and mechanisms of transposition, making them important putative actors in genome evolution. Additionally, LTR retrotransposons influence the gene expression patterns of adjacent genes by modulating potential small interfering RNA (siRNA) and RNA-directed DNA methylation (RdDM) pathways. Furthermore, comparative and evolutionary analysis of the most important crop genome sequences and advanced technologies have elucidated the epigenetics and structural and functional modifications driven by LTR retrotransposon during speciation. However, mechanistic insights into LTR retrotransposons remain obscure in plant development due to a lack of advancement in high throughput technologies. In this review, we focus on the key role of LTR retrotransposons response in plants during heat stress, the role of centromeric LTR retrotransposons, and the role of LTR retrotransposon markers in genome expression and evolution.

Correction: Ramakrishnan et al. The Dynamism of Transposon Methylation for Plant Development and Stress Adaptation. Int. J. Mol. Sci. 2021, 22, 11387.

The authors would like to make the following corrections to the original publication [...].

Artificial optimization of bamboo Ppmar2 transposase and host factors effects on Ppmar2 transposition in yeast.

Mariner-like elements (MLEs) are promising tools for gene cloning, gene expression, and gene tagging. We have characterized two MLE transposons from moso bamboo, Ppmar1 and Ppmar2. Ppmar2, is smaller in size and has higher natural activities, thus making it a more potential genomic tool compared to Ppmar1. Using a two-component system consisting of a transposase expression cassette and a non-autonomous transposon cotransformed in yeast, we investigated the transposition activity of Ppmar2 and created hyperactive transposases. Five out of 19 amino acid mutations in Ppmar2 outperformed the wild-type in terms of catalytic activities, especially with the S347R mutant having 6.7-fold higher transposition activity. Moreover, 36 yeast mutants with single-gene deletion were chosen to screen the effects of the host factors on Ppmar2NA transposition. Compared to the control strain (his3Δ), the mobility of Ppmar2 was greatly increased in 9 mutants and dramatically decreased in 7 mutants. The transposition ability in the efm1Δ mutant was 15-fold higher than in the control, while it was lowered to 1/66 in the rtt10Δ mutant. Transcriptomic analysis exhibited that EFM1 defection led to the significantly impaired DDR2, HSP70 expression and dramatically boosted JEN1 expression, whereas RTT10 defection resulted in significantly suppressed expression of UTP20, RPA190 and RRP5. Protein methylation, chromatin and RNA transcription may affect the Ppmar2NA transposition efficiency in yeast. Overall, the findings provided evidence for transposition regulation and offered an alternative genomic tool for moso bamboo and other plants.

The pattern of DNA methylation alteration, and its association with the expression changes of non-coding RNAs and mRNAs in Moso bamboo under abiotic stress.

Epigenetic changes play an important role in plant growth and development and in stress response. However, DNA methylation pattern and its relationship with the expression changes of non-coding RNAs and mRNAs of Moso bamboo in response to abiotic stress is still largely unknown. In this work, we used whole-genome bisulfite sequencing in combination with whole-transcriptome sequencing to analyze the DNA methylation and transcription patterns of mRNAs and non-coding RNAs in Moso bamboo under abiotic stresses such as cold, heat, ultraviolet (UV) and salinity. We found that CHH methylation in the promoter region was positively correlated with gene expression, while CHG and CHH methylations in the gene body regions were negatively associated with gene expression. Moreover, CG and CHG methylations in the promoter regions were negatively correlated with the transcript abundance of long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and circular RNAs (circRNAs). Similarly, the methylation levels of three contexts in the genic regions were negatively correlated with the transcript abundance of lncRNAs and miRNAs but positively correlated with that of circRNAs. In addition, we suggested that the reduction of 21-nt and 24-nt small interfering RNA (siRNA) expression tended to increase methylation levels in the genic regions. We found that stress-responsive genes such as CRPK1, HSFB2A and CIPK were differentially methylated and expressed. Our results also proposed that DNA methylation may regulate the expression of the transcription factors (TFs) and plant hormone signalling genes such as IAA9, MYC2 and ERF110 in response to abiotic stress. This study firstly reports the abiotic stress-responsive DNA methylation pattern and its involvement of expression of coding RNAs and non-coding RNAs in Moso bamboo. The results expand the knowledge of epigenetic mechanisms in Moso bamboo under abiotic stress and support in-depth deciphering of the function of specific non-coding RNAs in future studies.

Redox status of the plant cell determines epigenetic modifications under abiotic stress conditions and during developmental processes.

BACKGROUND: The oxidation-reduction (redox) status of the cell influences or regulates transcription factors and enzymes involved in epigenetic changes, such as DNA methylation, histone protein modifications, and chromatin structure and remodeling. These changes are crucial regulators of chromatin architecture, leading to differential gene expression in eukaryotes. But the cell's redox homeostasis is difficult to sustain since the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) is not equal in plants at different developmental stages and under abiotic stress conditions. Exceeding optimum ROS and RNS levels leads to oxidative stress and thus alters the redox status of the cell. Consequently, this alteration modulates intracellular epigenetic modifications that either mitigate or mediate the plant growth and stress response. AIM OF REVIEW: Recent studies suggest that the altered redox status of the cell reform the cellular functions and epigenetic changes. Recent high-throughput techniques have also greatly advanced redox-mediated gene expression discovery, but the integrated view of the redox status, and its associations with epigenetic changes and subsequent gene expression in plants are still scarce. In this review, we accordingly focus on how the redox status of the cell affects epigenetic modifications in plants under abiotic stress conditions and during developmental processes. This is a first comprehensive review on the redox status of the cell covering the redox components and signaling, redox status alters the post-translational modification of proteins, intracellular epigenetic modifications, redox interplay during DNA methylation, redox regulation of histone acetylation and methylation, redox regulation of miRNA biogenesis, redox regulation of chromatin structure and remodeling and conclusion, future perspectives and biotechnological opportunities for the future development of the plants. KEY SCIENTIFIC CONCEPTS OF REVIEW: The interaction of redox mediators such as ROS, RNS and antioxidants regulates redox homeostasis and redox-mediated epigenetic changes. We discuss how redox mediators modulate epigenetic changes and show the opportunities for smart use of the redox status of the cell in plant development and abiotic stress adaptation. However, how a redox mediator triggers epigenetic modification without activating other redox mediators remains yet unknown.

The plant epitranscriptome: revisiting pseudouridine and 2'-O-methyl RNA modifications.

There is growing evidence that post-transcriptional RNA modifications are highly dynamic and can be used to improve crop production. Although more than 172 unique types of RNA modifications have been identified throughout the kingdom of life, we are yet to leverage upon the understanding to optimize RNA modifications in crops to improve productivity. The contributions of internal mRNA modifications such as N6-methyladenosine (m6 A) and 5-methylcytosine (m5 C) methylations to embryonic development, root development, leaf morphogenesis, flowering, fruit ripening and stress response are sufficiently known, but the roles of the two most abundant RNA modifications, pseudouridine (Ψ) and 2'-O-methylation (Nm), in the cell remain unclear due to insufficient advances in high-throughput technologies in plant development. Therefore, in this review, we discuss the latest methods and insights gained in mapping internal Ψ and Nm and their unique properties in plants and other organisms. In addition, we discuss the limitations that remain in high-throughput technologies for qualitative and quantitative mapping of these RNA modifications and highlight future challenges in regulating the plant epitranscriptome.

Long terminal repeats (LTR) and transcription factors regulate PHRE1 and PHRE2 activity in Moso bamboo under heat stress.

BACKGROUND: LTR retrotransposons play a significant role in plant growth, genome evolution, and environmental stress response, but their regulatory response to heat stress remains unclear. We have investigated the activities of two LTR retrotransposons, PHRE1 and PHRE2, of moso bamboo (Phyllostachys edulis) in response to heat stress. RESULTS: The differential overexpression of PHRE1 and PHRE2 with or without CaMV35s promoter showed enhanced expression under heat stress in transgenic plants. The transcriptional activity studies showed an increase in transposition activity and copy number among moso bamboo wild type and Arabidopsis transgenic plants under heat stress. Comparison of promoter activity in transgenic plants indicated that 5'LTR promoter activity was higher than CaMV35s promoter. Additionally, yeast one-hybrid (Y1H) system and in planta biomolecular fluorescence complementation (BiFC) assay revealed interactions of heat-dependent transcription factors (TFs) with 5'LTR sequence and direct interactions of TFs with pol and gag. CONCLUSIONS: Our results conclude that the 5'LTR acts as a promoter and could regulate the LTR retrotransposons in moso bamboo under heat stress.

The Dynamism of Transposon Methylation for Plant Development and Stress Adaptation.

Plant development processes are regulated by epigenetic alterations that shape nuclear structure, gene expression, and phenotypic plasticity; these alterations can provide the plant with protection from environmental stresses. During plant growth and development, these processes play a significant role in regulating gene expression to remodel chromatin structure. These epigenetic alterations are mainly regulated by transposable elements (TEs) whose abundance in plant genomes results in their interaction with genomes. Thus, TEs are the main source of epigenetic changes and form a substantial part of the plant genome. Furthermore, TEs can be activated under stress conditions, and activated elements cause mutagenic effects and substantial genetic variability. This introduces novel gene functions and structural variation in the insertion sites and primarily contributes to epigenetic modifications. Altogether, these modifications indirectly or directly provide the ability to withstand environmental stresses. In recent years, many studies have shown that TE methylation plays a major role in the evolution of the plant genome through epigenetic process that regulate gene imprinting, thereby upholding genome stability. The induced genetic rearrangements and insertions of mobile genetic elements in regions of active euchromatin contribute to genome alteration, leading to genomic stress. These TE-mediated epigenetic modifications lead to phenotypic diversity, genetic variation, and environmental stress tolerance. Thus, TE methylation is essential for plant evolution and stress adaptation, and TEs hold a relevant military position in the plant genome. High-throughput techniques have greatly advanced the understanding of TE-mediated gene expression and its associations with genome methylation and suggest that controlled mobilization of TEs could be used for crop breeding. However, development application in this area has been limited, and an integrated view of TE function and subsequent processes is lacking. In this review, we explore the enormous diversity and likely functions of the TE repertoire in adaptive evolution and discuss some recent examples of how TEs impact gene expression in plant development and stress adaptation.

Bamboo Transposon Research: Current Status and Perspectives.

Bamboo, a fast-growing non-timber forest plant with many uses, is a valuable species for green development. However, bamboo flowering is very infrequent, extending, in general, for up to 120 years. Ecologically, bamboo species are generally better adapted to various environments than other grasses. Therefore, the species deserves a special status in what could be called Ecological Bioeconomy. An understanding of the genetic processes of bamboo can help us sustainably develop and manage bamboo forests. Transposable elements (TEs), jumping genes or transposons, are major genetic elements in plant genomes. The rapid development of the bamboo reference genome, at the chromosome level, reveals that TEs occupy over 63.24% of the genome. This is higher than found in rice, Brachypodium, and sorghum. The bamboo genome contains diverse families of TEs, which play a significant role in bamboo's biological processes including growth and development. TEs provide important clues for understanding the evolution of the bamboo genome. In this chapter, we briefly describe the current status of research on TEs in the bamboo genome, their regulation, and transposition mechanisms. Perspectives for future research are also provided.

Multi-omics analysis of cellular pathways involved in different rapid growth stages of moso bamboo.

Moso bamboo (Phyllostachys edulis (Carriere) J. Houzeau) is a rapidly growing grass of industrial and ecological importance. However, the molecular mechanisms of its remarkable growth are not well understood. In this study, we investigated the early-stage growth of moso bamboo shoots and defined three different growth stages based on histological and biochemical analyses, namely, starting of cell division (SD), rapid division (RD) and rapid elongation (RE). Further analyses on potentially relevant cellular pathways in these growth stages using multi-omics approaches such as transcriptomics and proteomics revealed the involvement of multiple cellular pathways, including DNA replication, repair and ribosome biogenesis. A total of 8045 differentially expressed genes (DEGs) and 1053 differentially expressed proteins (DEPs) were identified in our analyses. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses of detected DEGs identified several key biological pathways such as phytohormone metabolism, signal transduction, cell wall development and carbohydrate metabolism. The comparative analysis of proteins displayed that a total of 213 DEPs corresponded with DEGs and 3 significant expression profiles that could be promoting the fast growth of bamboo internodes. Moreover, protein-protein interaction network prediction analysis is suggestive of the involvement of five major proteins of signal transduction, DNA synthesis and RNA transcription, and may act as key elements responsible for the rapid shoot growth. Our work exploits multi-omics and bioinformatic approaches to unfurl the complexity of molecular networks involved in the rapid growth of moso bamboo and opens up questions related to the interactions between the functions played by individual molecular pathway.

Nuclear export signal (NES) of transposases affects the transposition activity of mariner-like elements Ppmar1 and Ppmar2 of moso bamboo.

Ppmar1 and Ppmar2 are two active mariner-like elements (MLEs) cloned from moso bamboo (Phyllostachys edulis (Carrière) J. Houz) genome possessing transposases that harbour nuclear export signal (NES) domain, but not any nuclear localization signal (NLS) domain. To understand the functions of NES in transposon activity, we have conducted two experiments, fluorescence and excision frequency assays in the yeast system. For this, by site-directed mutagenesis, three NES mutants were developed from each of the MLE. In the fluorescence assay, the mutants, NES-1, 2 and 3 along with the wild types (NES-0) were fused with fluorescent proteins, enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) were co-transformed into yeast system. To differentiate protein localisation under the NES influence, ECFP alone was fused to wild and mutant NES domains either on N- or C-terminal and not to EYFP. Fluorescence assay revealed that blue fluorescence of ECFP was more intense than the red fluorescence of the EYFP in the yeast cell matrix. Further, ECFP had a wider localisation in the cellular matrix, but EYFP was largely located in the nucleus. The NES-1 domain was related to the comparatively high spread of ECFP, while NES-2 and NES-3 indicated a low spread, implying that NES activity on nuclear export increased when the NES is made leucine-rich, while the signalling activity was reduced when the leucine content was lowered in the NES domain. In the transposon excision assay, the mutant and wild type NES of both the Ppmar elements were integrated into an Ade2 vector, and within the Ade2 gene. Co-transformation of the vector together with non-autonomous Ppmar transposons and NES-lacking transposases was used to assess the differential excision frequencies of the mutants NES domains. In both the MLEs, NES-1 had the highest excision suppression, which was less than half of the excision frequency of the wild type. NES-2 and NES-3 elements showed, up to three times increase in transposon excision than the wild types. The results suggested that NES is an important regulator of nuclear export of transposase in Ppmar elements and the mutation of the NES domains can either increase or decrease the export signalling. We speculate that in moso bamboo, NESs regulates the transposition activity of MLEs to maintain the genome integrity.

Affinities of Terminal Inverted Repeats to DNA Binding Domain of Transposase Affect the Transposition Activity of Bamboo Ppmar2 Mariner-Like Element.

Mariner-like elements (MLE) are a super-family of DNA transposons widespread in animal and plant genomes. Based on their transposition characteristics, such as random insertions and high-frequency heterogeneous transpositions, several MLEs have been developed to be used as tools in gene tagging and gene therapy. Two active MLEs, Ppmar1 and Ppmar2, have previously been identified in moso bamboo (Phyllostachys edulis). Both of these have a preferential insertion affinity to AT-rich region and their insertion sites are close to random in the host genome. In Ppmar2 element, we studied the affinities of terminal inverted repeats (TIRs) to DNA binding domain (DBD) and their influence on the transposition activity. We could identify two putative boxes in the TIRs which play a significant role in defining the TIR's affinities to the DBD. Seven mutated TIRs were constructed, differing in affinities based on similarities with those of other plant MLEs. Gel mobility shift assays showed that the TIR mutants with mutation sites G669A-C671A had significantly higher affinities than the mutants with mutation sites C657T-A660T. The high-affinity TIRs indicated that their transposition frequency was 1.5-2.0 times higher than that of the wild type TIRs in yeast transposition assays. The MLE mutants with low-affinity TIRs had relatively lower transposition frequency from that of wild types. We conclude that TIR affinity to DBD significantly affects the transposition activity of Ppmar2. The mutant MLEs highly active TIRs constructed in this study can be used as a tool for bamboo genetic studies.