Transcriptome and microRNA Sequencing Identified miRNAs and Target Genes in Different Developmental Stages of the Vascular Cambium in Cryptomeria fortunei Hooibrenk

Cryptomeria fortunei Hooibrenk is an important fast-growing coniferous timber species that is widely used in landscaping. Recently, research on timber quality has gained substantial attention in the field of tree breeding. Wood is the secondary xylem formed by the continuous inward division and differentiation of the vascular cambium; therefore, the development of the vascular cambium is particularly important for wood quality. In this study, we analyzed the transcriptomes of the cambial zone in C. fortunei during different developmental stages using Illumina HiSeq sequencing, focusing on general transcriptome and microRNA (miRNA) data. We performed functional annotation of the differentially expressed genes (DEGs) in the different stages identified by transcriptome sequencing and generated 15 miRNA libraries yielding 4.73 Gb of clean reads. The most common length of the filtered miRNAs was 21nt, accounting for 33.1% of the total filtered reads. We annotated a total of 32 known miRNA families. Some miRNAs played roles in hormone signal transduction (miR159, miR160, and miR166), growth and development (miR166 and miR396), and the coercion response (miR394 and miR395), and degradome sequencing showed potential cleavage sites between miRNAs and target genes. Differential expression of miRNAs and target genes and functional validation of the obtained transcriptome and miRNA data provide a theoretical basis for further elucidating the molecular mechanisms of cellular growth and differentiation, as well as wood formation in the vascular cambium, which will help improve the wood quality of C. fortunei.

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Wood Transcriptome Profiling Identifies Critical Pathway Genes of Secondary Wall Biosynthesis and Novel Regulators for Vascular Cambium Development in Populus

Genes ◽

10.3390/genes10090690 ◽

2019 ◽

Vol 10 (9) ◽

pp. 690 ◽

Cited By ~ 1

Author(s):

Min-Ha Kim ◽

Jin-Seong Cho ◽

Hyung-Woo Jeon ◽

Kanidta Sangsawang ◽

Donghwan Shim ◽

...

Keyword(s):

Secondary Wall ◽

Molecular Mechanisms ◽

Developmental Stages ◽

Hybrid Poplar ◽

Secondary Growth ◽

Leaf Tissue ◽

Transcriptome Profiling ◽

Wood Formation ◽

Vascular Cambium ◽

Critical Pathway

Wood, the most abundant biomass on Earth, is composed of secondary xylem differentiated from vascular cambium. However, the underlying molecular mechanisms of wood formation remain largely unclear. To gain insight into wood formation, we performed a series of wood-forming tissue-specific transcriptome analyses from a hybrid poplar (Populus alba × P. glandulosa, clone BH) using RNA-seq. Together with shoot apex and leaf tissue, cambium and xylem tissues were isolated from vertical stem segments representing a gradient of secondary growth developmental stages (i.e., immature, intermediate, and mature stem). In a comparative transcriptome analysis of the ‘developing xylem’ and ‘leaf’ tissue, we could identify critical players catalyzing each biosynthetic step of secondary wall components (e.g., cellulose, xylan, and lignin). Several candidate genes involved in the initiation of vascular cambium formation were found via a co-expression network analysis using abundantly expressed genes in the ‘intermediate stem-derived cambium’ tissue. We found that transgenic Arabidopsis plants overexpressing the PtrHAM4-1, a GRAS family transcription factor, resulted in a significant increase of vascular cambium development. This phenotype was successfully reproduced in the transgenic poplars overexpressing the PtrHAM4-1. Taken together, our results may serve as a springboard for further research to unravel the molecular mechanism of wood formation, one of the most important biological processes on this planet.

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Cloning and Functional Analysis of Lignin Biosynthesis Genes Cf4CL and CfCCoAOMT in Cryptomeria fortunei

Genes ◽

10.3390/genes10080619 ◽

2019 ◽

Vol 10 (8) ◽

pp. 619 ◽

Cited By ~ 2

Author(s):

Zhenhao Guo ◽

Hui Hua ◽

Jin Xu ◽

Jiaxing Mo ◽

Hui Zhao ◽

...

Keyword(s):

Gene Editing ◽

Bioinformatics Analysis ◽

Southern China ◽

Lignin Biosynthesis ◽

Wood Formation ◽

Primary Component ◽

Vascular Cambium ◽

Timber Species ◽

Wood Production ◽

Lignin Synthesis

Cryptomeria fortunei, also known as the Chinese cedar, is an important timber species in southern China. The primary component of its woody tissues is lignin, mainly present in secondary cell walls. Therefore, continuous lignin synthesis is crucial for wood formation. In this study, we aimed to discover key genes involved in lignin synthesis expressed in the vascular cambium of C. fortunei. Through transcriptome sequencing, we detected expression of two genes, 4CL and CCoAOMT, known to be homologous to enzymes involved in the lignin synthesis pathway. We studied the function of these genes through bioinformatics analysis, cloning, vascular cambium expression analysis, and transgenic cross-species functional validation studies. Our results show that Cf4CL and CfCCoAOMT do indeed function in the pathway of lignin synthesis and likely perform this function in C. fortunei. They are prime candidates for future (gene-editing) studies aimed at optimizing C. fortunei wood production.

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Integrated analysis of sex-biased mRNA and miRNA expression profiles in the gonad of the discus fish (Symphysodon aequifasciatus)

10.1101/492264 ◽

2018 ◽

Cited By ~ 1

Author(s):

yuanshuai Fu ◽

Zhe Xu ◽

Zaizhong Chen ◽

Bin Wen ◽

Jianzhong Gao

Keyword(s):

Molecular Mechanisms ◽

Target Genes ◽

High Efficiency ◽

Expression Profiles ◽

Gonad Development ◽

Differentially Expressed ◽

Cdna Libraries ◽

Integrated Analysis ◽

Illumina Hiseq ◽

Differentially Expressed Mirnas

The discus fish (Symphysodon aequifasciatus) is an ornamental fish that is well-known around the world. Phenotype investigation indicated that there are no significant differences in appearance between males and females of the discus fish. To better understand the sexual development mechanisms and obtain a high efficiency sex identification method in the artificial reproduction process of the discus fish, we constructed six cDNA libraries from three adult testes and three adult ovaries, and perform RNA-sequencing for identifying sex-biased candidate genes, microRNA (miRNA), and metabolic pathway using the Illumina Hiseq 4000. A total of 50,082 non-redundant genes (unigenes) were identified, of which 18,570 unigenes were significantly overexpressed in testes, and 11,182 unigenes were significantly overexpressed in ovaries, and 8 differentially expressed unigenes were validated by quantitative Real-Time PCR (qPCR). A total of 551 miRNAs were identified, of which 47 miRNAs were differentially expressed between testes and ovaries, and 7 differentially expressed miRNAs and one non-differential miRNA were validated by qPCR. Twenty-four of these differentially expressed miRNAs and their 15 predicted target genes constituted 41 important miRNA-mRNA interaction pairs, which may be important candidates for sex-related miRNAs and sex-related genes in the discus fish. Some of vital sex-related metabolic pathways were also identified that may play key roles in regulating gonad development of the discus fish. These results can provide important insights to better understand molecular mechanisms for sexual dimorphism in gonads development.

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Identification and validation of key miRNAs and miRNA–mRNA regulatory network associated with uterine involution in postpartum Kazakh sheep

Archives Animal Breeding ◽

10.5194/aab-64-119-2021 ◽

2021 ◽

Vol 64 (1) ◽

pp. 119-129

Author(s):

Heng Yang ◽

Lin Fu ◽

Qifeng Luo ◽

Licai Li ◽

Fangling Zheng ◽

...

Keyword(s):

Mirna Target ◽

Molecular Mechanisms ◽

Target Genes ◽

Gene Prediction ◽

Biological Effects ◽

Differentially Expressed ◽

Illumina Hiseq ◽

Go Annotation ◽

Uterine Involution ◽

Differentially Expressed Mirnas

Abstract. MicroRNAs (miRNAs) are widely expressed in different mammalian tissues and exert their biological effects through corresponding target genes. miRNA target genes can be rapidly and efficiently identified and screened by combining bioinformatics prediction and experimental validation. To investigate the possible molecular regulatory mechanisms involving miRNAs during uterine involution in postpartum ewes, we used Illumina HiSeq sequencing technology to screen for the number and characteristics of miRNAs in faster uterine involution and normal uterine involution group. A total of 118 differentially expressed miRNAs, including 33 known miRNAs and 85 new miRNAs, were identified in the hypothalamic library, whereas 54 miRNAs, including 5 known miRNAs and 49 new miRNAs, were identified in the uterine library. Screening with four types of gene prediction software revealed 73 target genes associated with uterine involution, and subsequently, GO annotation and KEGG pathway analysis were performed. The results showed that, in the hypothalamic–uterine axis, uterine involution in postpartum ewes might primarily involve two miRNA-target gene pairs, namely, miRNA-200a–PTEN and miRNA-133–FGFR1, which can participate in GnRH signal transduction in the upstream hypothalamus and in the remodeling process at the downstream uterus, through the PI3K–AKT signaling pathway to influence the recovery of the morphology and functions of the uterus during the postpartum period in sheep. Therefore, identification of differentially expressed miRNAs in this study fills a gap in the research related to miRNAs in uterine involution in postpartum ewes and provides an important reference point for a comprehensive understanding of the molecular mechanisms underlying the regulation of postpartum uterine involution in female livestock.

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Analysis of Transcriptome and miRNAome in the Muscle of Bamei Pigs at Different Developmental Stages

Animals ◽

10.3390/ani10071198 ◽

2020 ◽

Vol 10 (7) ◽

pp. 1198 ◽

Cited By ~ 2

Author(s):

Guofang Wu ◽

Lin Ma ◽

Lei Wang ◽

Jiping Zhou ◽

Yuhong Ma ◽

...

Keyword(s):

Skeletal Muscle ◽

Meat Quality ◽

Molecular Mechanisms ◽

Target Genes ◽

Developmental Stages ◽

Enrichment Analysis ◽

Rna Seq ◽

Developmental Processes ◽

Citrate Cycle ◽

Molecular Approaches

The growth of skeletal muscle involves complex developmental processes that play an important part in the determinization of pork quality. The investigation of skeletal muscle mRNA or miRNA profiles is especially important for finding molecular approaches to improve meat quality in pig breeding. Therefore, we studied the transcriptome (mRNA and miRNA) profiles of skeletal muscle with RNA-Seq in three developmental stages of pigs: 65-day embryonic (E65), postnatal 0 days (natal) and 10 months (adult). We found 10,035, 9050 and 4841 differentially expressed (DE) genes for natal vs. E65, adult vs. E65 and adult vs. natal, 55, 101 and 85 DE miRNA for natal vs. E65, adult vs. E65 and adult vs. natal, respectively. In addition, the target genes of DE miRNA that was in a negative correlation with the corresponding miRNA in the same comparison group were selected for enrichment analysis. Gene Ontology terms were mainly classified into developmental processes. Pathway analysis revealed enrichment in the Rap1 signaling pathway, citrate cycle and oxidative phosphorylation and carbon. Finally, RT-PCR was employed for validating the level of expression of 11 DE miRNA and 14 DEGs. The transcriptome profiles of skeletal muscle from the different developmental stages of the Bamei pigs were obtained. From these data, hundreds of DE miRNA and mRNA, and the miRNA–mRNA regulatory network can provide valuable insights into further understanding of key molecular mechanisms and improving the meat quality in pig breeding.

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Genome-Wide Identification of Long Non-Coding RNAs and Their Potential Functions in Poplar Growth and Phenylalanine Biosynthesis

Frontiers in Genetics ◽

10.3389/fgene.2021.762678 ◽

2021 ◽

Vol 12 ◽

Author(s):

Lei Zhang ◽

Xiaolan Ge ◽

Jiujun Du ◽

Xingqi Cheng ◽

Xiaopeng Peng ◽

...

Keyword(s):

Target Genes ◽

Expression Patterns ◽

Wood Quality ◽

Target Prediction ◽

Wood Formation ◽

Sequencing Analysis ◽

Oxidoreductase Activity ◽

Regulatory Processes ◽

Genome Wide ◽

Phenylpropanoid Biosynthesis

Poplar is an important bioenergy tree species. lncRNAs play important roles in various biological regulatory processes, and their expression pattern is more tissue-specific than mRNAs. In this study, P. deltoides “Danhong” (Pd) and P. simonii “Tongliao1” (Ps) with different growth rates and wood quality were used as experimental materials, and the transcriptomes of their shoot apical meristem, xylem, and phloem were sequenced. Furthermore, high-throughput RNA sequencing analysis revealed that the expression patterns of genes and lncRNAs are different between the two genotypes. 6,355 lncRNAs were identified. Based on target prediction, lncRNAs and target genes were involved in ADP binding, oxidoreductase activity, phenylpropanoid biosynthesis, and cyanoamino acid metabolism. The DElncRNAs in two poplars were co-expressed with transcription factors and structural genes of lignin and flavonoid pathways. In addition, we found the potential target lncRNAs of miRNA. This result provides basic evidence for a better understanding of the regulatory role of lncRNAs in regulating phenylalanine molecular pathways and wood formation.

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The Cytoskeleton and Its Role in Determining Cellulose Microfibril Angle in Secondary Cell Walls of Woody Tree Species

Plants ◽

10.3390/plants9010090 ◽

2020 ◽

Vol 9 (1) ◽

pp. 90

Author(s):

Tobias ◽

Spokevicius ◽

McFarlane ◽

Bossinger

Keyword(s):

Molecular Mechanisms ◽

Cellulose Microfibril ◽

Wood Quality ◽

Microfibril Angle ◽

Wood Formation ◽

Cellulose Microfibrils ◽

Reaction Wood ◽

Molecular Control ◽

Cellulose Deposition ◽

Cellulose Microfibril Angle

Recent advances in our understanding of the molecular control of secondary cell wall (SCW) formation have shed light on molecular mechanisms that underpin domestication traits related to wood formation. One such trait is the cellulose microfibril angle (MFA), an important wood quality determinant that varies along tree developmental phases and in response to gravitational stimulus. The cytoskeleton, mainly composed of microtubules and actin filaments, collectively contribute to plant growth and development by participating in several cellular processes, including cellulose deposition. Studies in Arabidopsis have significantly aided our understanding of the roles of microtubules in xylem cell development during which correct SCW deposition and patterning are essential to provide structural support and allow for water transport. In contrast, studies relating to SCW formation in xylary elements performed in woody trees remain elusive. In combination, the data reviewed here suggest that the cytoskeleton plays important roles in determining the exact sites of cellulose deposition, overall SCW patterning and more specifically, the alignment and orientation of cellulose microfibrils. By relating the reviewed evidence to the process of wood formation, we present a model of microtubule participation in determining MFA in woody trees forming reaction wood (RW).

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Comparative Analyses of Full-Length Transcriptomes Reveal Gnetum luofuense Stem Developmental Dynamics

Frontiers in Genetics ◽

10.3389/fgene.2021.615284 ◽

2021 ◽

Vol 12 ◽

Author(s):

Chen Hou ◽

Huiming Lian ◽

Yanling Cai ◽

Yingli Wang ◽

Dongcheng Liang ◽

...

Keyword(s):

Molecular Mechanisms ◽

Target Genes ◽

Developmental Stages ◽

Secondary Growth ◽

Alternative Polyadenylation ◽

Cost Effective ◽

Developmental Trajectory ◽

Full Length ◽

Dynamic Pattern ◽

Primary Growth

Genus Gnetum, of which the majority species are pantropical liana, have broad industrial uses including for string, nets, and paper production. Although numerous studies have investigated anatomical structures during stem development, the underlying molecular mechanisms that regulate this developmental trajectory in Gnetum species remain poorly understood. A total of 12 full-length transcriptomes were generated from four stem developmental stages of an arborescent representative of this genus, Gnetum luofuense, using Oxford Nanopore Technologies. The results of this analysis reveal a total of 24,151 alternative splicing (AS) and 134,391 alternative polyadenylation events. A remarkably dynamic pattern of AS events, especially in the case of intron retentions, was found across the four developmental stages while no dynamic pattern was found among transcript numbers with varied poly(A) sites. A total of 728 long non-coding RNAs were also detected; the number of cis-regulated target genes dramatically increased while no changes were found among trans-regulated target genes. In addition, a K-means clustering analysis of all full-length transcripts revealed that primary growth is associated with carbohydrate metabolism and fungi defense, while secondary growth is closely linked with photosynthesis, nitrogen transportation, and leaf ontogenesis. The use of weighted gene co-expression network analysis as well as differentially expressed transcripts reveals that bHLH, GRF, and MYB-related transcription factors are involved in primary growth, while AP2/ERF, MYB, NAC, PLAZ, and bZIP participate in G. luofuense stem secondary growth. The results of this study provide further evidence that Nanopore sequencing technology provides a cost-effective method for generating full-length transcriptome data as well as for investigating seed plant organ development.

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Identification and Tissue-Specific Characterization of Novel SHOX-Regulated Genes in Zebrafish Highlights SOX Family Members Among Other Genes

Frontiers in Genetics ◽

10.3389/fgene.2021.688808 ◽

2021 ◽

Vol 12 ◽

Author(s):

Sandra Hoffmann ◽

Ralph Roeth ◽

Sabrina Diebold ◽

Jasmin Gogel ◽

David Hassel ◽

...

Keyword(s):

Short Stature ◽

Molecular Mechanisms ◽

Target Genes ◽

Human Fibroblasts ◽

Family Members ◽

Cellular Growth ◽

Whole Body ◽

Growth Disorders ◽

Tissue Specific ◽

Pectoral Fins

SHOX deficiency causes a spectrum of clinical phenotypes related to skeletal dysplasia and short stature, including Léri-Weill dyschondrosteosis, Langer mesomelic dysplasia, Turner syndrome, and idiopathic short stature. SHOX controls chondrocyte proliferation and differentiation, bone maturation, and cellular growth arrest and apoptosis via transcriptional regulation of its direct target genes NPPB, FGFR3, and CTGF. However, our understanding of SHOX-related pathways is still incomplete. To elucidate the underlying molecular mechanisms and to better understand the broad phenotypic spectrum of SHOX deficiency, we aimed to identify novel SHOX targets. We analyzed differentially expressed genes in SHOX-overexpressing human fibroblasts (NHDF), and confirmed the known SHOX target genes NPPB and FGFR among the most strongly regulated genes, together with 143 novel candidates. Altogether, 23 genes were selected for further validation, first by whole-body characterization in developing shox-deficient zebrafish embryos, followed by tissue-specific expression analysis in three shox-expressing zebrafish tissues: head (including brain, pharyngeal arches, eye, and olfactory epithelium), heart, and pectoral fins. Most genes were physiologically relevant in the pectoral fins, while only few genes were also significantly regulated in head and heart tissue. Interestingly, multiple sox family members (sox5, sox6, sox8, and sox18) were significantly dysregulated in shox-deficient pectoral fins together with other genes (nppa, nppc, cdkn1a, cdkn1ca, cyp26b1, and cy26c1), highlighting an important role for these genes in shox-related growth disorders. Network-based analysis integrating data from the Ingenuity pathways revealed that most of these genes act in a common network. Our results provide novel insights into the genetic pathways and molecular events leading to the clinical manifestation of SHOX deficiency.

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Transcriptome-wide identification and characterization of microRNAs in diverse phases of wood formation in Populus trichocarpa

10.21203/rs.3.rs-108245/v1 ◽

2020 ◽

Author(s):

Ruiqi Wang ◽

Mengxuan Ren ◽

Shuanghui Tian ◽

Cong Liu ◽

He Cheng ◽

...

Keyword(s):

Cell Wall ◽

Target Genes ◽

Developmental Stages ◽

Populus Trichocarpa ◽

Wood Formation ◽

Pcr Analysis ◽

Integrated Analysis ◽

Cell Wall Modification ◽

Dynamic Regulation ◽

Genome Wide

Abstract Background: MicroRNAs (miRNAs) are small, non-coding RNAs that have important regulatory functions in plant growth and development. However, the miRNAs that are involved in different developmental stages of tree stems have not been systemically characterized. In this study, we applied miRNA expression profiling method to the Populus trichocarpa trunks of the three distinct developmental stages defined as the primary stem (PS), transitional stem (TS), and secondary stem (SS) to investigate the miRNA species, their dynamic regulation and functions during the transitions of wood formation in different developmental stages at the genome-wide scale by Solexa sequencing.Results: We obtained 892, 872, and 882 known miRNAs and 1,727, 1,723, and 1,597 novel miRNAs, from PS, TS, and SS, respectively. And identified 114, 306, and 152 differentially expressed miRNAs (DE-miRNAs) with 921, 2,639, and 2,042 candidate target genes (CTGs), which formed 158, 855, and 297 DE-miRNA-CTG pairs in PS vs TS, PS vs SS, and TS vs SS , respectively. Among these, 47, 439, and 71 DE-miRNA-CTG pairs showed a significant negative correlation, respectively. Finally, we identified 39, 9, and 92 miRNA-CTG pairs involved in PS, TS, and SS, respectively. These DE-miRNA-CTG pairs in poplar or whose counterparts in other plant species are known to be transcriptional factors or structural genes involved in cell division and differentiation, cell wall modification, secondary cell wall (SCW) biosynthesis, lignification, and programmed cell death processes of wood formation. Moreover, qRT–PCR analysis confirmed that the results of small RNA-seq were robust and reliable and most miRNA-CTG pairs exhibited an inverse correlation.Conclusions: This is the first report on an integrated analysis of genome-wide mRNA and miRNA profiling of diverse phases of wood formation in poplar trunks. We showed that even though miRNAs involved in diverse developmental phases were not in a considerable number, their roles in the regulatory network that govern wood formation during different developmental stages cannot be negligible or underestimated. The information and data obtained in this paper significantly advanced our understanding of these miRNAs and their essential, dynamic and diversified roles as well as functions in diverse phases of wood formation in tree species.

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