Implantation of the blastocyst into the uterus is the gateway for further embryonic development in mammals. Programming of blastocyst to an implantation-competent state known as blastocyst activation ...is the determining factor for implantation into the receptive uterus. However, it remains largely unclear how the blastocyst is globally programmed for implantation. Employing a delayed implantation mouse model, we show here that the blastocyst undergoes extensive programming essential for implantation. By analyzing the transcriptional profile of blastocysts with different implantation competency, we reveal the dynamic change in the biosynthesis, metabolism, and proliferation during blastocyst reactivation from diapause. We also demonstrate that reactivation of the X chromosome, one of the most important events during periimplantation of female embryonic development, is not completed even in blastocysts under conditions of dormancy, despite long term suspension in the uterus. Moreover, the mural trophectoderm (TE), but not the polar TE, differentiates to be more invasive through the weakened cell-cell tight junctions and extracellular matrices (ECMs). By analyzing the differentially expressed profile of secretory proteins, we further demonstrate that the blastocyst functions as a proinflammatory body to secrete proinflammatory signals, such as TNFα and S100A9, thereby triggering embryouterine attachment reaction during implantation. Collectively, our data systematically and comprehensively disclose the programming of blastocyst reactivation from diapause for implantation and uncover previously undefined roles of blastocyst during implantation.
Nanopore sequencing from Oxford Nanopore Technologies (ONT) and Pacific BioSciences (PacBio) single-molecule real-time (SMRT) long-read isoform sequencing (Iso-Seq) are revolutionizing the way ...transcriptomes are analyzed. These methods offer many advantages over most widely used high-throughput short-read RNA sequencing (RNA-Seq) approaches and allow a comprehensive analysis of transcriptomes in identifying full-length splice isoforms and several other post-transcriptional events. In addition, direct RNA-Seq provides valuable information about RNA modifications, which are lost during the PCR amplification step in other methods. Here, we present a comprehensive summary of important applications of these technologies in plants, including identification of complex alternative splicing (AS), full-length splice variants, fusion transcripts, and alternative polyadenylation (APA) events. Furthermore, we discuss the impact of the newly developed nanopore direct RNA-Seq in advancing epitranscriptome research in plants. Additionally, we summarize computational tools for identifying and quantifying full-length isoforms and other co/post-transcriptional events and discussed some of the limitations with these methods. Sequencing of transcriptomes using these new single-molecule long-read methods will unravel many aspects of transcriptome complexity in unprecedented ways as compared to previous short-read sequencing approaches. Analysis of plant transcriptomes with these new powerful methods that require minimum sample processing is likely to become the norm and is expected to uncover novel co/post-transcriptional gene regulatory mechanisms that control biological outcomes during plant development and in response to various stresses.
Auxin is essential for plant growth and development. Although substantial progress has been made in understanding auxin pathways in model plants such as Arabidopsis and rice, little is known in moso ...bamboo which is famous for its fast growth resulting from the rapid cell elongation and division.
Here we showed that exogenous auxin has strong effects on crown and primary roots. Genes involved in auxin action, including 13 YUCCA (YUC) genes involved in auxin synthesis, 14 PIN-FORMED/PIN-like (PIN/PILS) and 7 AUXIN1/LIKE-AUX1 (AUX1/LAX) members involved in auxin transport, 10 auxin receptors (AFB) involved in auxin perception, 43 auxin/indole-3-aceticacid (AUX/IAA) genes, and 41 auxin response factors (ARF) involved in auxin signaling were identified through genome-wide analysis. Phylogenetic analysis of these genes from Arabidopsis, Oryza sativa and bamboo revealed that auxin biosynthesis, transport, and signaling pathways are conserved in these species. A comprehensive study of auxin-responsive genes using RNA sequencing technology was performed, and the results also supported that moso bamboo shared a conserved regulatory mechanism for the expression of auxin pathway genes; meanwhile it harbors its own specific properties.
In summary, we generated an overview of the auxin pathway in bamboo, which provides information for uncovering the precise roles of auxin pathway in this important species in the future.
Alternative splicing (AS) is a major post‐transcriptional mechanism to enhance the diversity of proteome in response to environmental signals. Among the numerous external signals perceived by plants, ...light is the most crucial one. Plants utilize complex photoreceptor signaling networks to sense different light conditions and adjust their growth and development accordingly. Although light‐mediated gene expression has been widely investigated, little is known regarding the mechanism of light affecting AS to modulate mRNA at the post‐transcriptional level. In this minireview, we summarize current progresses on how light affects AS, and how sensory photoreceptors and retrograde signaling pathways may coordinately regulate AS of pre‐mRNAs. In addition, we also discuss the possibility that AS of the mRNAs encoding photoreceptors may be involved in feedback control of AS. We hypothesize that light regulation of the expression and activity of splicing factors would be a major mechanism of light‐mediated AS. The combination of genetic study and high‐throughput analyses of AS and splicing complexes in response to light is likely to further advance our understanding of the molecular mechanisms underlying light control of AS and plant development.
In plant, light affects alternative splicing mainly via three primary types of sensory photoreceptor pathways or retrograde signals from chloroplast, while photoreceptors themselves are also under the regulation of alternative splicing. The light regulation of splicing factors would be a major mechanism of light‐mediated alternative splicing that represents functions on dominant negative regulation, subcellular localization, or taking part in nonsense‐mediated mRNA decay pathway.
Summary
In mammals, DNA methylation is associated with aging. However, age‐related DNA methylation changes during phase transitions largely remain unstudied in plants. Moso bamboo (Phyllostachys ...edulis) requires a very long time to transition from the vegetative to the floral phase. To comprehensively investigate the association of DNA methylation with aging, we present here single‐base‐resolution DNA methylation profiles using both high‐throughput bisulfite sequencing and single‐molecule nanopore‐based DNA sequencing, covering the long period of vegetative growth and transition to flowering in moso bamboo. We discovered that CHH methylation gradually accumulates from vegetative to reproductive growth in a time‐dependent fashion. Differentially methylated regions, correlating with chronological aging, occurred preferentially at both transcription start sites and transcription termination sites. Genes with CG methylation changes showed an enrichment of Gene Ontology (GO) categories in ‘vegetative to reproductive phase transition of meristem’. Combining methylation data with mRNA sequencing revealed that DNA methylation in promoters, introns and exons may have different roles in regulating gene expression. Finally, circular RNA (circRNA) sequencing revealed that the flanking introns of circRNAs are hypermethylated and enriched in long terminal repeat (LTR) retrotransposons. Together, the observations in this study provide insights into the dynamic DNA methylation and circRNA landscapes, correlating with chronological age, which paves the way to study further the impact of epigenetic factors on flowering in moso bamboo.
Significance Statement
Moso bamboo requires a very long time to transition from the vegetative to the floral phase. Here, we found that the DNA methylation level varies with chronological age in moso bamboo. This study also provides insights into flanking introns of circRNAs, which are hypermethylated and enriched in LTR retrotransposons.
The nucleosome assembly protein 1 (NAP1) family is the main histone chaperone of histone H2A–H2B. To explore the function of NAP1 family genes in moso bamboo (Phyllostachys edulis), characterized by ...extremely rapid growth and a long flowering cycle, we originally conducted a genome-wide analysis of the PheNAP1 gene. The phylogenetic relationship, gene expression pattern, DNA methylation, and histone modification were analyzed. Eventually, 12 PheNAP1 genes were recognized from the Phyllostachys edulis genome, divided into two sorts: the NRP subfamily (four members) and the NAP subfamily (eight members). Highly conserved motifs exist in each subfamily, which are distinct between subfamilies. PheNAP1 was distributed homogeneously on 10 out of 24 chromosomes, and gene duplication contributed significantly to the enhancement of the PheNAP1 gene in the genome. Cis-acting element analysis showed that PheNAP1 family genes are involved in light, hormone, and abiotic stress responses and may play an important role in the rapid growth and flowering. PheNAP1 exhibited the highest expression level in fast-growing shoots, indicating it is closely associated with the rapid growth of moso bamboo. Besides, PheNAP1 can rescue the early-flowering phenotype of nrp1-1 nrp2-2, and it affected the expression of genes related to the flowering pathway, like BSU1, suggesting the vital role that PheNAP1 may take in the flowering process of moso bamboo. In addition, histone modification results showed that PheNAP1 could bind to phosphorylation-, acetylation-, and methylation-modified histones to further regulate gene expression. A sketch appears: that PheNAP1 can accompany histones to regulate fast-growth- and flowering-related genes in moso bamboo. The consequences of this study enrich the understanding of the epigenetic regulation mechanism of bamboo plants and lays a foundation for further studies on the role of the NAP1 gene in Phyllostachys edulis and the function of chromatin regulation in forest growth and development.
Moso bamboo (Phyllostachys edulis) is a well-known bamboo species of high economic value in the textile industry due to its rapid growth. Phytohormones, which are master regulators of growth and ...development, serve as important endogenous signals. However, the mechanisms through which phytohormones regulate growth in moso bamboo remain unknown to date.
Here, we reported that exogenous gibberellins (GA) applications resulted in a significantly increased internode length and lignin condensation. Transcriptome sequencing revealed that photosynthesis-related genes were enriched in the GA-repressed gene class, which was consistent with the decrease in leaf chlorophyll concentrations and the lower rate of photosynthesis following GA treatment. Exogenous GA applications on seedlings are relatively easy to perform, thus we used 4-week-old whole seedlings of bamboo for GA- treatment followed by high throughput sequencing. In this study, we identified 932 cis-nature antisense transcripts (cis-NATs), and 22,196 alternative splicing (AS) events in total. Among them, 42 cis-nature antisense transcripts (cis-NATs) and 442 AS events were differentially expressed upon exposure to exogenous GA
, suggesting that post-transcriptional regulation might be also involved in the GA
response. Targets of differential expression of cis-NATs included genes involved in hormone receptor, photosynthesis and cell wall biogenesis. For example, LAC4 and its corresponding cis-NATs were GA
-induced, and may be involved in the accumulation of lignin, thus affecting cell wall composition.
This study provides novel insights illustrating how GA alters post-transcriptional regulation and will shed light on the underlying mechanism of growth modulated by GA in moso bamboo.
Dendrocalamus latiflorus Munro is a woody clumping bamboo with rapid shoot growth. Both genetic transformation and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated ...protein 9 (Cas9) gene editing techniques are available for D. latiflorus, enabling reverse genetic approaches. Thus, D. latiflorus has the potential to be a model bamboo species. However, the genome sequence of D. latiflorus has remained unreported due to its polyploidy and large genome size. Here, we sequenced the D. latiflorus genome and assembled it into three allele‐aware subgenomes (AABBCC), representing the largest genome of a major bamboo species. We assembled 70 allelic chromosomes (2, 737 Mb) for hexaploid D. latiflorus using both single‐molecule sequencing from the Pacific Biosciences (PacBio) Sequel platform and chromosome conformation capture sequencing (Hi‐C). Repetitive sequences comprised 52.65% of the D. latiflorus genome. We annotated 135 231 protein‐coding genes in the genome based on transcriptomes from eight different tissues. Transcriptome sequencing using RNA‐Seq and PacBio single‐molecule real‐time long‐read isoform sequencing revealed highly differential alternative splicing (AS) between non‐abortive and abortive shoots, suggesting that AS regulates the abortion rate of bamboo shoots. This high‐quality hexaploid genome and comprehensive strand‐specific transcriptome datasets for this Poaceae family member will pave the way for bamboo research using D. latiflorus as a model species.
The allele‐aware chromosome‐scale assembly of the genome of the hexaploid Ma bamboo Dendrocalamus latiflorus by Pacific Biosciences Sequel sequencing and chromosome conformation capture sequencing will facilitate overexpression and CRISPRCas9 gene editing studies.
ABSTRACT
Bamboo is one of the fastest growing plants among monocotyledonous species and is grown extensively in subtropical regions. Although bamboo has high economic value and produces much biomass ...quickly, gene functional research is hindered by the low efficiency of genetic transformation in this species. We therefore explored the potential of a bamboo mosaic virus (BaMV)‐mediated expression system to investigate genotype–phenotype associations. We determined that the sites between the triple gene block proteins (TGBps) and the coat protein (CP) of BaMV are the most efficient insertion sites for the expression of exogenous genes in both monopodial and sympodial bamboo species. Moreover, we validated this system by individually overexpressing the two endogenous genes ACE1 and DEC1, which resulted in the promotion and suppression of internode elongation, respectively. In particular, this system was able to drive the expression of three 2A‐linked betalain biosynthesis genes (more than 4 kb in length) to produce betalain, indicating that it has high cargo capacity and may provide the prerequisite basis for the development of a DNA‐free bamboo genome editing platform in the future. Since BaMV can infect multiple bamboo species, we anticipate that the system described in this study will greatly contribute to gene function research and further promote the molecular breeding of bamboo.
Bamboo mosaic virus (BaMV) shows high cargo capacity and this virus‐mediated expression system could be used to investigate genotype–phenotype associations in monopodial and sympodial bamboo.
(
) belongs to Gymnospermae, which are fast-growing and have desirable wood properties. However,
's stress resistance is little understood. To unravel the physiological and molecular regulation ...mechanisms under environmental stresses in the typical gymnosperm species of
, three-year-old plants were exposed to simulated drought stress (polyethylene glycol 8000), salicylic acid, and cold treatment at 4 °C for 8 h, 32 h, and 56 h, respectively. Regarding the physiological traits, we observed a decreased protein content and increased peroxidase upon salicylic acid and polyethylene glycol treatment. Superoxide dismutase activity either decreased or increased at first and then returned to normal under the stresses. Regarding the molecular regulation, we used both nanopore direct RNA sequencing and short-read sequencing to reveal a total of 5646 differentially expressed genes in response to different stresses, of which most had functions in lignin catabolism, pectin catabolism, and xylan metabolism, indicating that the development of stem-differentiating xylem was affected upon stress treatment. Finally, we identified a total of 51 AP2/ERF, 29 NAC, and 37 WRKY transcript factors in
. The expression of most of the NAC TFs increased under cold stress, and the expression of most of the WRKY TFs increased under cold and SA stress. These results revealed the transcriptomics responses in
to short-term stresses under this study's experimental conditions and provide preliminary clues about stem-differentiating xylem changes associated with different stresses.
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