Genomic survey and expression analysis of LcARFs reveal multiple functions to somatic embryogenesis in Liriodendron
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ABSTRACT:
SUBMITTER: Xu L
PROVIDER: S-EPMC10848544 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
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Project description:The differential expression of genes is crucial for plant somatic embryogenesis (SE), and the accurate quantification of gene expression levels relies on choosing appropriate reference genes. To select the most suitable reference genes for SE studies, 10 commonly used reference genes were examined in synchronized somatic embryogenic and subsequent germinative cultures of Liriodendron hybrids by using quantitative real-time reverse transcription PCR. Four popular normalization algorithms: geNorm, NormFinder, Bestkeeper and Delta-Ct were used to select and validate the suitable reference genes. The results showed that elongation factor 1-gamma, histone H1 linker protein, glyceraldehyde-3-phosphate dehydrogenase and α-tubulin were suitable for SE tissues, while elongation factor 1-gamma and actin were best for the germinative organ tissues. Our work will benefit future studies of gene expression and functional analyses of SE in Liriodendron hybrids. It is also serves as a guide of reference gene selection in early embryonic gene expression analyses for other woody plant species.
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Project description:AT-hook motif nuclear localized (AHL) is a transcription factor that can directly induce plant somatic embryogenesis without adding exogenous hormones. One of its functional domains, the AT-hook motif, has a chromatin-modifying function and participates in various cellular processes, including DNA replication and repair and gene transcription leading to cell growth. Liriodendron chinense (Hemsl.) Sargent is an important ornamental and timber tree in China. However, its low drought-resistant ability further leads to a low natural growth rate of its population. Based on bioinformatics analysis, this study identified a total of 21 LcAHLs in L. chinense. To explore the expression pattern of the AHL gene family under drought and somatic embryogenesis, we performed a systematic analysis including basic characteristics, gene structure, chromosome localization, replication event, cis-acting elements and phylogenetic analyses. According to the phylogenetic tree, the 21 LcAHL genes are divided into three separate clades (Clade I, II, and III). Cis-acting element analysis indicated the involvement of the LcAHL genes in drought, cold, light, and auxin regulation. In the generated drought stress transcriptome, a total of eight LcAHL genes showed increased expression levels, with their expression peaking at 3 h and leveling off after 1 d. Nearly all LcAHL genes were highly expressed in the process of somatic embryogenesis. In this study, we performed a genome-wide analysis of the LcAHL gene family and found that LcAHLs take part in resistance to drought stress and the development of somatic embryos. These findings will provide an important theoretical basis for understanding of the LcAHL gene function.
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Project description:The basic leucine zip (bZIP) transcription factors (TFs) are a group of highly conserved gene families that play important roles in plant growth and resistance to adversity stress. However, studies on hormonal regulatory pathways and functional analysis during somatic embryogenesis (SE) in Dimocarpus longan is still unavailable. In this study, a total of 51 bZIP family members were systematically identified in the whole genome of longan, a comprehensive bioinformatics analysis of DlbZIP (bZIP family members of D. longan) was performed, and subcellular localization and profiles patterns after transiently transformed DlbZIP60 were analyzed. The combined analysis of RNA-seq, ATAC-seq and ChIP-seq showed that four members have different H3K4me1 binding peaks in early SE and differentially expressed with increased chromatin accessibility. Comparative transcriptome analysis of bZIPs expression in early SE, different tissues and under 2,4-D treatment revealed that DlbZIP family might involved in growth and development during longan early SE. The qRT-PCR results implied that DlbZIP family were subjected to multiple hormonal responses and showed different degrees of up-regulated expression under indole-3-acetic acid (IAA), abscisic acid (ABA) and methyl jasmonate (MeJA) treatments, which indicated that they played an important role in the hormone synthesis pathways associated with the early SE of longan. Subcellular localization showed that DlbZIP60 was located in the nucleus, and the contents of endogenous IAA, MeJA and ABA were up-regulated in transiently DlbZIP60 overexpressed cell lines. These results suggest that DlbZIP60 may mediate hormones pathways that functions the development during early SE in longan.
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Project description:Expression and function of the transcription factor Sox10 is predominant in neural crest cells, its derivatives and in oligodendrocytes. To understand how Sox10 expression is regulated during development, we analysed the potential of evolutionary conserved non-coding sequences in the Sox10 genomic region to function as enhancers. By linking these sequences to a beta-galactosidase marker gene under the control of a minimal promoter, five regulatory regions were identified that direct marker gene expression in transgenic mice to Sox10 expressing cell types and tissues in a defined temporal pattern. These possible enhancers of the Sox10 gene mediate Sox10 expression in the otic vesicle, in oligodendrocytes and in several neural crest derivatives including the developing peripheral nervous system and the adrenal gland. They furthermore exhibit overlapping activities and share binding sites for Sox, Lef/Tcf, Pax and AP2 transcription factors. This may explain high level and robustness of Sox10 expression during embryonic development.
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Project description:In this study, 52 AAAP genes were identified in the L. chinense genome and divided into eight subgroups based on phylogenetic relationships, gene structure, and conserved motif. A total of 48 LcAAAP genes were located on the 14 chromosomes, and the remaining four genes were mapped in the contigs. Multispecies phylogenetic tree and codon usage bias analysis show that the LcAAAP gene family is closer to the AAAP of Amborella trichopoda, indicating that the LcAAAP gene family is relatively primitive in angiosperms. Gene duplication events revealed six pairs of segmental duplications and one pair of tandem duplications, in which many paralogous genes diverged in function before monocotyledonous and dicotyledonous plants differentiation and were strongly purification selected. Gene expression pattern analysis showed that the LcAAAP gene plays a certain role in the development of Liriodendron nectary and somatic embryogenesis. Low temperature, drought, and heat stresses may activate some WRKY/MYB transcription factors to positively regulate the expression of LcAAAP genes to achieve long-distance transport of amino acids in plants to resist the unfavorable external environment. In addition, the GAT and PorT subgroups could involve gamma-aminobutyric acid (GABA) transport under aluminum poisoning. These findings could lay a solid foundation for further study of the biological role of LcAAAP and improvement of the stress resistance of Liriodendron.
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Project description:BackgroundEthylene production and signalling play an important role in somatic embryogenesis, especially for species that are recalcitrant in in vitro culture. The AP2/ERF superfamily has been identified and classified in Hevea brasiliensis. This superfamily includes the ERFs involved in response to ethylene. The relative transcript abundance of ethylene biosynthesis genes and of AP2/ERF genes was analysed during somatic embryogenesis for callus lines with different regeneration potential, in order to identify genes regulated during that process.ResultsThe analysis of relative transcript abundance was carried out by real-time RT-PCR for 142 genes. The transcripts of ERFs from group I, VII and VIII were abundant at all stages of the somatic embryogenesis process. Forty genetic expression markers for callus regeneration capacity were identified. Fourteen markers were found for proliferating calli and 35 markers for calli at the end of the embryogenesis induction phase. Sixteen markers discriminated between normal and abnormal embryos and, lastly, there were 36 markers of conversion into plantlets. A phylogenetic analysis comparing the sequences of the AP2 domains of Hevea and Arabidopsis genes enabled us to predict the function of 13 expression marker genes.ConclusionsThis first characterization of the AP2/ERF superfamily in Hevea revealed dramatic regulation of the expression of AP2/ERF genes during the somatic embryogenesis process. The gene expression markers of proliferating callus capacity to regenerate plants by somatic embryogenesis should make it possible to predict callus lines suitable to be used for multiplication. Further functional characterization of these markers opens up prospects for discovering specific AP2/ERF functions in the Hevea species for which somatic embryogenesis is difficult.
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Project description:MicroRNAs (miRNAs) are endogenous non-coding ~21 nucleotide RNAs that regulate gene expression at the transcriptional and post-transcriptional levels in plants and animals. They play an important role in development, abiotic stress, and pathogen responses. miRNAs with their targets have been widely studied in model plants, but limited knowledge is available on the small RNA population of cotton (Gossypium hirsutum)-an important economic crop, and global identification of related targets through degradome sequencing has not been developed previously. In this study, small RNAs and their targets were identified during cotton somatic embryogenesis (SE) through high-throughput small RNA and degradome sequencing, comparing seedling hypocotyl and embryogenic callus (EC) of G. hirsutum YZ1. A total of 36 known miRNA families were found to be differentially expressed, of which 19 miRNA families were represented by 29 precursors. Twenty-five novel miRNAs were identified. A total of 234 transcripts in EC and 322 transcripts in control (CK) were found to be the targets of 23 and 30 known miRNA families, respectively, and 16 transcripts were targeted by eight novel miRNAs. Interestingly, four trans-acting small interfering RNAs (tas3-siRNAs) were also found in degradome libraries, three of which perfectly matched their precursors. Several targets were further validated via RNA ligase-mediated rapid amplification of 5' cDNA ends (RLM 5'-RACE). The profiling of the miRNAs and their target genes provides new information on the miRNAs network during cotton SE.
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Project description:The Sm proteins are a conserved protein family with Sm motifs. The family includes Sm and Sm-like proteins, which play important roles in pre-mRNA splicing. Most research on the Sm proteins have been conducted in herbaceous plants, and less in woody plants such as Dimocarpus longan (longan). And the embryo development status significantly affects the quality and yield of longan. In this study, we conducted a genome-wide analysis of longan Sm genes (DlSm) to clarify their roles during somatic embryogenesis (SE) and identified 29 Sm genes. Phylogenetic analysis deduced longan Sm proteins clustered into 17 phylogenetic groups with the homologous Sm proteins of Arabidopsis thaliana. We also analyzed the gene structures, motif compositions, and conserved domains of the longan Sm proteins. The promoter sequences of the DlSm genes contained many light, endosperm development, hormone, and temperature response elements, which suggested their possible functions. In the non-embryogenic callus(NEC) and during early SE in longan, the alternative splicing(AS) events of DlSm genes indicated that these genes may influence SE development by changing gene structures and sequences. The kinetin(KT) hormone, and blue and white light treatments affected the differentiation and growth of longan embryonic callus(EC) probably by affecting the AS events of DlSm genes. Expression profiles showed the possible functional divergence among Sm genes, and different hormones and light qualities affected their expression levels. The expression trends of the DlSm genes determined by RNA sequencing as fragments per kilobase of exon model per million mapped reads (FPKM) and by real-time quantitative PCR(qRT-PCR) during early SE in longan showed that the expression of the DlSm genes was affected by the growth and differentiation of longan SE, and decreased as the somatic embryo differentiation progressed. The results will contributed to understanding the longan Sm gene family and provide a basis for future functional validation studies.
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Project description:Plant somatic embryos are widely used in the fields of germplasm conservation, breeding for genetic engineering and artificial seed production. MicroRNAs (miRNAs) play pivotal roles in somatic embryogenesis (SE) regulation. However, their regulatory roles during various stages of SE remain unclear. In this study, six types of embryogenic samples of Lilium pumilum DC. Fisch., including organogenic callus, embryogenic callus induced for 4 weeks, embryogenic callus induced for 6 weeks, globular embryos, torpedo embryos and cotyledon embryos, were prepared for small RNA sequencing. The results revealed a total of 2,378,760 small RNA reads, among which the most common size was 24 nt. Four hundred and fifty-two known miRNAs, belonging to more than 86 families, 57 novel miRNAs and 40 miRNA*s were identified. The 86 known miRNA families were sorted according to an alignment with their homologs across 24 land plants into the following four categories: 23 highly conserved, 4 moderately conserved, 15 less conserved and 44 species-specific miRNAs. Differentially expressed known miRNAs were identified during various stages of SE. Subsequently, the expression levels of 12 differentially expressed miRNAs and 4 targets were validated using qRT-PCR. In addition, six samples were mixed in equal amounts for transcript sequencing, and the sequencing data were used as transcripts for miRNA target prediction. A total of 66,422 unigenes with an average length of 800 bp were assembled from 56,258,974 raw reads. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment indicated that 38,004 and 15,497 unigenes were successfully assigned to GO terms and KEGG pathways, respectively. Among the unigenes, 2,182 transcripts were predicted to be targets for 396 known miRNAs. The potential targets of the identified miRNAs were mostly classified into the following GO terms: cell, binding and metabolic process. Enriched KEGG analysis demonstrated that carbohydrate metabolism was the predominant pathway in Lilium SE. Thus, we performed systemic characterization, homology comparisons and profiling of miRNA expression, and we constructed an miRNA-target network during Lilium SE for the first time. Our findings establish a foundation for the further exploration of critical genes and elucidation of SE in Lilium.
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