Degradome Sequencing Research Articles

Identification of miR160 family and their target genes in Rehmannia glutinosa in response to endophytic fungus GG22 infection

This study aims to reveal the possible role of miR160 family in Rehmannia glutinosa in response to the infection of endophytic fungus Fusarium oxysporum GG22. Specifically, miR160 precursors and mature miR160 were retrieved from the small RNA database yielded by high-throughput sequencing. RNAfold was used to analyze the precursor structure, and DNAMAN and MEGA to analyze conservation and evolution of miR160 precursors and mature miR160. The target genes of miR160 were predicted and annotated, and the interaction was analyzed. Based on degradome sequencing, the target genes were further identified. The results showed that miR160 precursors had intact stem-loop structures. The precursor and mature sequences were conserved, particularly the 3 rd-16 th bases of the 5'-terminal. According to the phylogenetic tree, R. glutinosa had close evolutionary relationship with Arabidopsis thaliana, Oryza sativa, Salvia miltiorrhiza, and Sesamum indicum. A total of 22 target genes of miR160 were predicted and most of them were auxin response factor(ARF) genes. The target genes were involved in the Gene Ontology(GO) terms of biological processes, cellular components, and molecular functions. According to the degradome sequencing results, four target genes of miR160 were ARF(ARF18, ARF22) genes. R. glutinosa regulated its growth in response to the infection of endophytic fungus by changing the expression of miR160 and the target genes. qRT-PCR result of the differentially expressed rgl-miR160a and rgl-miR160a-3p was consistent with the sequencing result. This study clarifies the molecular mechanism of R. glutinosa in response to GG22 stress, laying a theoretical basis for the improvement and future research of R. glutinosa.

Degradome sequencing reveals an integrative miRNA-mediated gene interaction network regulating rice seed vigor

BackgroundIt is well known that seed vigor is essential for agricultural production and rice (Oryza sativa L.) is one of the most important crops in the world. Though we previously reported that miR164c regulates rice seed vigor, but whether and how other miRNAs cooperate with miR164c to regulate seed vigor is still unknown.ResultsBased on degradome data of six RNA samples isolated from seeds of the wild-type (WT) indica rice cultivar ‘Kasalath’ as well as two modified lines in ‘Kasalath’ background (miR164c-silenced line [MIM164c] and miR164c overexpression line [OE164c]), which were subjected to either no aging treatment or an 8-day artificial aging treatment, 1247 different target transcripts potentially cleaved by 421 miRNAs were identified. The miRNA target genes were functionally annotated via GO and KEGG enrichment analyses. By STRING database assay, a miRNA-mediated gene interaction network regulating seed vigor in rice was revealed, which comprised at least four interconnected pathways: the miR5075-mediated oxidoreductase related pathway, the plant hormone related pathway, the miR164e related pathway, and the previously reported RPS27AA related pathway. Knockout and overexpression of the target gene Os02g0817500 of miR5075 decreased and enhanced seed vigor, respectively. By Y2H assay, the proteins encoded by five seed vigor-related genes, Os08g0295100, Os07g0633100, REFA1, OsPER1 and OsGAPC3, were identified to interact with Os02g0817500.ConclusionsmiRNAs cooperate to regulate seed vigor in rice via an integrative gene interaction network comprising miRNA target genes and other functional genes. The result provided a basis for fully understanding the molecular mechanisms of seed vigor regulation.

Integrate Small RNA and Degradome Sequencing to Reveal Drought Memory Response in Wheat (Triticum aestivum L.).

Drought has gradually become one of the most severe abiotic stresses on plants. Plants that experience stress training can exhibit enhanced stress tolerance. According to MicroRNA (miRNA) sequencing data, this study identified 195 candidate drought memory-related miRNAs in wheat, and targets of 64 (32.8%) candidate miRNAs were validated by degradome sequencing. Several drought memory-related miRNAs such as tae-miR9676-5p, tae-MIR9676-p3_1ss21GA, tae-miR171a, tae-miR531_L-2, tae-miR408_L-1, PC-3p-5049_3565, tae-miR396c-5p, tae-miR9778, tae-miR164a-5p, and tae-miR9662a-3p were validated as having a strong response to drought memory by regulating the expression of their target genes. In addition, overexpression of drought memory-related miRNA, tae-miR531_L-2, can remarkably improve the drought tolerance of transgenic Arabidopsis thaliana. Drought memory can regulate plant cellular signal transduction, plant biosynthetic processes, and other biological processes to cope with drought via transcriptional memory. In addition, drought memory-related miRNAs can promote starch and sucrose catabolism and soluble sugar accumulation and regulate proline homeostasis to improve plant drought resistance. Our results could contribute to an understanding of drought memory in wheat seedlings and may provide a new strategy for drought-resistant breeding.

Integrated Expression Analysis of Small RNA, Degradome and Microarray Reveals Complex Regulatory Action of miRNA during Prolonged Shade in Swarnaprabha Rice.

Simple SummarySignificant progress has been made to understand shade and low-light due to their deteriorative impact on plant growth and crop yield. The authors of the present study identified novel microRNA (miR) regulations for tolerant phenotypic responses associated with a sustainable yield of Swarnaprabha, a shade-tolerant rice genotype. We present three new findings, two of which are demonstrations of novel concepts. First, we show that the differentially expressed miRs had drastic differences in the category of targets based on their function and pathway. Second, we show that neutrally regulated and uniquely expressed miRs can also contribute to the shade-tolerance response by altering the differential expression of their targets under different light conditions. Third, we identified the interactions of 16 miRNA and 21 target pairs, whose actions can significantly contribute to the shade-tolerance phenotype and sustainable yield of Swarnaprabha rice. The findings of this study will significantly update the knowledge of the epigenetic regulations of shade tolerance in rice. The authors of further studies can use the genomic data and findings of this study to generate shade-tolerant crop varieties to combat prevailing unpredictable weather conditions in order to maintain food security.Prolonged shade during the reproductive stage can result in significant yield losses in rice. For this study, we elucidated the role of microRNAs in prolonged-shade tolerance (~20 days of shade) in a shade-tolerant rice variety, Swarnaprabha (SP), in its reproductive stage using small RNA and degradome sequencing with expression analysis using microarray and qRT-PCR. This study demonstrates that miRNA (miR) regulation for shade-tolerance predominately comprises the deactivation of the miR itself, leading to the upregulation of their targets. Up- and downregulated differentially expressed miRs (DEms) presented drastic differences in the category of targets based on the function and pathway in which they are involved. Moreover, neutrally regulated and uniquely expressed miRs also contributed to the shade-tolerance response by altering the differential expression of their targets, probably due to their differential binding affinities. The upregulated DEms mostly targeted the cell wall, membrane, cytoskeleton, and cellulose synthesis-related transcripts, and the downregulated DEms targeted the transcripts of photosynthesis, carbon and sugar metabolism, energy metabolism, and amino acid and protein metabolism. We identified 16 miRNAs with 21 target pairs, whose actions may significantly contribute to the shade-tolerance phenotype and sustainable yield of SP. The most notable among these were found to be miR5493-OsSLAC and miR5144-OsLOG1 for enhanced panicle size, miR5493-OsBRITTLE1-1 for grain formation, miR6245-OsCsIF9 for decreased stem mechanical strength, miR5487-OsGns9 and miR168b-OsCP1 for better pollen development, and miR172b-OsbHLH153 for hyponasty under shade.

The Construction and Exploration of a Comprehensive MicroRNA Centered Regulatory Network in Foxtail Millet (Setaria italica L.).

MicroRNA (miRNA) is an essential endogenous post-transcriptional regulatory factor, and foxtail millet (Setaria italica L.) is an ideal C4 model cereal that is a highly valuable crop in semiarid and arid areas. The Research on comprehensive and high confidence identification and annotation of foxtail millet miRNAs needs to be strengthened, and to our knowledge, there is no information on the regulatory network of foxtail millet miRNA. In this study, 136 high confidence miRNAs were identified through high-throughput sequencing of the small RNAs in seven tissues at the shooting and grain filling stages of foxtail millet. A total of 2,417 target genes were obtained by combining computational biology software and degradome sequencing methods. Furthermore, an analysis using transcriptome sequencing revealed the relationships between miRNAs and their target genes and simultaneously explored key regulatory modules in panicles during the grain filling stage. An miRNA regulatory network was constructed to explore the functions of miRNA in more detail. This network, centered on miRNAs and combining upstream transcriptional factors and downstream target genes, is primarily composed of feed forward loop motifs, which greatly enhances our knowledge of the potential functions of miRNAs and uncovers numerous previously unknown regulatory links. This study provides a solid foundation for research on the function and regulatory network of miRNAs in foxtail millet.

Identification of anthocyanin-related microRNAs in ornamental kale (Brassica oleracea L. var. acephala) by high throughput sequencing

Anthocyanins have been found to be responsible for the pigmentation of red, purple and blue colors in plants. MicroRNA (miRNA) plays an important role in the regulation of anthocyanin biosynthesis in plants. However, few miRNAs and their corresponding functions related to anthocyanin biosynthesis in ornamental kale (Brassica oleracea var. acephala) have been reported. In this study, small RNA (sRNA) and degradome libraries from inner and outer leaves (IL and OL) of ornamental kale line “Pigeon Red” were constructed, respectively. Totally, 139 known and 14 novel miRNAs were identified by sRNA sequencing, and 304 target genes cleaved by 92 bo-miRNAs were identified by degradome sequencing. Of these, 5 miRNAs and their corresponding targets were potentially involved in the anthocyanin biosynthesis. And 35 miRNAs were differently expressed between IL and OL. Integrated analysis of sRNA-Seq, degradome sequencing and RNA-Seq revealed that bo-miR828 and its targets potentially play a key role in the regulation of anthocyanin biosynthesis in ornamental kale leaves. Finally, an inverse correlation between the expression of bo-miR828 and its targets was revealed by both qRT-PCR and high-throughput sequencing. These results represented a comprehensive expression profiling of miRNAs related to anthocyanin accumulation in B.oleracea var. acephala and provided important clues for understanding the regulatory network of anthocyanin biosynthesis mediated by miRNA in Brassica crops.

An organ-specific transcriptomic atlas of the medicinal plant Bletilla striata: Protein-coding genes, microRNAs, and regulatory networks.

As one of the important species belonging to the Bletilla genus of Orchidaceae, Bletilla striata (Thunb.) Rchb. f., possesses both ornamental and medicinal values. Its dried tubers are used as a traditional Chinese medicine, and several secondary metabolites have been indicated to be the active ingredients. However, the molecular mechanisms related to the regulation of secondary metabolism have not been characterized in B. striata. In this study, integrated analysis of RNA sequencing (RNA-seq), small RNA sequencing (sRNA-seq), and degradome sequencing (degradome-seq) data from three organs (leaf, root, and tuber) of B. striata provided us with a comprehensive view of the microRNA (miRNA)-mediated regulatory network. Firstly, based on the RNA-seq data, the organ-specific expression patterns of the protein-coding genes, especially for those related to secondary metabolism, were investigated. Secondly, 342 conserved miRNA candidates were identified from B. striata. These miRNAs were assigned to 88 families, some of which were selected for expression pattern analysis. Additionally, 31 hairpin-structured precursors encoding 23 novel miRNAs were uncovered from the transcriptome assembly. Thirdly, based on the degradome signatures, 1,142 validated miRNA-target pairs (involving 167 conserved miRNAs and six novel miRNAs and 51 target genes) were included in the regulatory network. Organ-specific expression level comparison between the miRNAs and their targets revealed some interesting miRNA-target pairs. Fourthly, some valuable subnetworks were extracted for further functional studies. Additionally, some regulatory pathways were indicated to be monocot specific. Summarily, our results lay a solid basis for in-depth studies on the regulatory mechanisms underlying the production of the medicinal ingredients in B. striata.

Integrated Degradome and Srna Sequencing Revealed miRNA-mRNA Regulatory Networks between the Phloem and Developing Xylem of Poplar.

Lignin and cellulose are the most abundant natural organic polymers in nature. MiRNAs are a class of regulatory RNAs discovered in mammals, plants, viruses, and bacteria. Studies have shown that miRNAs play a role in lignin and cellulose biosynthesis by targeting key enzymes. However, the specific miRNAs functioning in the phloem and developing xylem of Populus deltoides are still unknown. In this study, a total of 134 miRNAs were identified via high-throughput small RNA sequencing, including 132 known and two novel miRNAs, six of which were only expressed in the phloem. A total of 58 differentially expressed miRNAs (DEmiRNAs) were identified between the developing xylem and the phloem. Among these miRNAs, 21 were significantly upregulated in the developing xylem in contrast to the phloem and 37 were significantly downregulated. A total of 2431 target genes of 134 miRNAs were obtained via high-throughput degradome sequencing. Most target genes of these miRNAs were transcription factors, including AP2, ARF, bHLH, bZIP, GRAS, GRF, MYB, NAC, TCP, and WRKY genes. Furthermore, 13 and nine miRNAs were involved in lignin and cellulose biosynthesis, respectively, and we validated the miRNAs via qRT-PCR. Our study explores these miRNAs and their regulatory networks in the phloem and developing xylem of P. deltoides and provides new insight into wood formation.

Small RNA Sequencing Revealed that miR4415, a Legume-Specific miRNA, was Involved in the Cold Acclimation of Ammopiptanthus nanus by Targeting an L-Ascorbate Oxidase Gene and Regulating the Redox State of Apoplast.

MicroRNAs (miRNAs) are small endogenous single-stranded RNAs that regulate plant growth, development, and environmental stress response posttranscriptionally. Ammopiptanthus nanus, a rare evergreen broad-leaved shrub in the temperate area of Central Asia, can tolerate freezing stress as low as –30 degrees centigrade in winter, and miRNA might be involved in the cold acclimation which enables A. nanus to obtain tolerance to freezing stress. Systematic identification and functional analysis of the miRNAs involved in the cold acclimation in A. nanus may promote understanding of the miRNA-mediated gene regulation network underlying cold acclimation. Here, based on small RNA and degradome sequencing, 256 miRNAs and 1,808 miRNA-target pairs were identified in A. nanus. A total of 39 cold-responsive miRNAs were identified, of which 29 were upregulated and ten were downregulated. These cold-responsive miRNAs may participate in the cold acclimation by regulating redox homeostasis (miR398, miR4415, and miR408), calcium signaling (miR5225 and miR5211), growth and development (miR159 and miR390), and small RNA–mediated gene silencing (miR168 and miR1515). We found that miR4415, a legume-specific miRNA, is involved in the cold acclimation of A. nanus by targeting an L-ascorbate oxidase gene and then regulating the redox state of the apoplast. Our study provides important data for understanding the regulatory role of miRNA in the cold acclimation of A. nanus.

MicroRNA and Degradome Profiling Uncover Defense Response of Fraxinus velutina Torr. to Salt Stress.

Soil salinization is a major environmental problem that seriously threatens the sustainable development of regional ecosystems and local economies. Fraxinus velutina Torr. is an excellent salt-tolerant tree species, which is widely planted in the saline-alkaline soils in China. A growing body of evidence shows that microRNAs (miRNAs) play important roles in the defense response of plants to salt stress; however, how miRNAs in F. velutina exert anti-salt stress remains unclear. We previously identified two contrasting F. velutina cuttings clones, salt-tolerant (R7) and salt-sensitive (S4) and found that R7 exhibits higher salt tolerance than S4. To identify salt-responsive miRNAs and their target genes, the leaves and roots of R7 and S4 exposed to salt stress were subjected to miRNA and degradome sequencing analysis. The results showed that compared with S4, R7 showed 89 and 138 differentially expressed miRNAs in leaves and roots, respectively. Specifically, in R7 leaves, miR164d, miR171b/c, miR396a, and miR160g targeting NAC1, SCL22, GRF1, and ARF18, respectively, were involved in salt tolerance. In R7 roots, miR396a, miR156a/b, miR8175, miR319a/d, and miR393a targeting TGA2.3, SBP14, GR-RBP, TCP2/4, and TIR1, respectively, participated in salt stress responses. Taken together, the findings presented here revealed the key regulatory network of miRNAs in R7 responding to salt stress, thereby providing new insights into improving salt tolerance of F. velutina through miRNA manipulation.

Comparative Transcriptome Analysis Uncovers the Regulatory Roles of MicroRNAs Involved in Petal Color Change of Pink-Flowered Strawberry.

The pink-flowered strawberry is popular in China due to its high ornamental value. In the present study, sRNAome, transcriptome, and degradome sequencing were performed to understand the functions of microRNAs (miRNAs) and their target genes during flower development in pink-flowered strawberry. Nine small RNA libraries and a mixed degradome library from flower petals at different developmental stages were constructed and sequenced. A total of 739 known miRNAs and 964 novel miRNAs were identified via small RNA sequencing, and 639 miRNAs were identified to cleave 2,816 target genes based on the degradome data. Additionally, 317 differentially expressed miRNAs among the various stages of flower development were identified, which regulated 2,134 differentially expressed target genes. These target genes were significantly enriched in the transcriptional regulation, phenylpropanoid biosynthesis, and plant hormone signal transduction pathways. Furthermore, integrated microRNAomic and transcriptomic analyses suggested that 98 miRNAs targeted several transcription factors, including MYBs (26), bHLHs (12), NACs (14), and SPLs (19), related to anthocyanin accumulation. In addition, 27 differentially expressed miRNAs might affect anthocyanin biosynthesis by regulating 23 targets involved in the hormone signal transduction pathway. The quantitative real-time PCR (qRT-PCR) analysis confirmed the expression changes of 21 miRNA-target pairs. Furthermore, the transient expression of candidate miRNAs was performed in the pink-flowered strawberry cultivar “Fenyun” at the bud stage. Introduction of FamiR156a, FamiR396e, and FamiR858_R-2 in the “Fenyun” increased flower color intensity, while transient expression of FamiR828a decreased flower color intensity. Overall, the present study uncovers the regulatory functions of microRNAs, including anthocyanin biosynthesis, hormone signaling, and regulation factors during flower development and coloration in pink-flowered strawberry. This work expands the knowledge of miRNAs affecting coloration in strawberry and provides rich resources for future functional studies.

Transcriptional multiomics reveals the mechanism of seed deterioration in Nicotiana tabacum L. and Oryza sativa L.

IntroductionMature seeds deteriorate gradually and die eventually during long-term storage. Controlled deterioration is often used to accelerate the seed deterioration rate to assess the seed vigor and physiological quality of seed lots. ObjectivesAlthough it is well known that the process of seed deterioration produced by controlled deterioration is distinct from that caused by long-term storage, the differences in transcriptional levels have not been reported. Clarifying the mechanism of seed deterioration is critical for identifying, conserving and utilizing germplasm resources. MethodsTobacco (Nicotiana tabacum L.) seeds were studied thoroughly using transcriptome, small RNA, and degradome sequencing after long-term storage (LS) and controlled deterioration (CD). Co-expression trend analysis identified transcripts involved in tobacco seed deterioration, while phylogenetic analysis helped to uncover comparable targets in rice (Oryza sativa L.) for further verification and utilization. ResultsIn LS and CD, a total of 2,112 genes and 164 miRNAs were differentially expressed, including 20 interaction miRNA-mRNA pairs with contrasting expression. Transcriptional multiomics found that the main causes of LS were plant hormone signal transduction and protein processing in the endoplasmic reticulum, whereas the primary cause of CD was nucleotide excision repair dysfunction. The homeostatic balance of RNA degradation and the spliceosome occurred in both modes of seed deterioration. Additionally, co-expression trend analysis identified two coherent pairs, nta-miR160b-NtARF18 and nta-miR396c-NtMBD10, as being significant in LS and CD, respectively. For utilization, rice homologous targets OsARF18 and OsMBD707 were verified to play similar roles in LS and CD, respectively. ConclusionThis study demonstrated the transcriptional mechanism of tobacco and key genes in seed deterioration. And the application of key genes in rice also verified the feasibility of the multiomics method, guiding the identification of candidate genes to precisely delay seed deterioration in other species of seed research.

Integrated transcriptome, small RNA and degradome analysis provide insights into the transcriptional regulatory networks underlying cold acclimation in jojoba

Jojoba (Simmondsia chinensis) is an emerging industrial oil crop cultivated in desert dry land. Jojoba is sensitive to cold stress, which hinders its domestication in the arid areas of temperate climate regions. Here, the effects of cold acclimation on physiological parameters and gene expression in jojoba leaves were analyzed. The tolerance to cold stress in cold-acclimated jojoba seedlings was enhanced as revealed by delayed fluorescence and photosynthetic parameter measurement. The expression levels of 7520 genes were changed significantly after cold acclimation, and these genes were related to multiple biological processes including the stability of photosynthetic machinery, the enhancement of ROS scavenging ability, and protein folding and RNA processing. A total of 144 miRNAs were identified in jojoba, including 133 conserved miRNAs and 11 species-specific miRNAs, and 106 targets with high confidence of miRNA were identified by degradome sequencing, including 18 novel targets of the conserved miRNAs. Forty-two miRNAs were differentially expressed in jojoba under cold acclimation, and these miRNAs were involved in cold acclimation by negatively regulating their targets. Integrated analysis revealed that multiple gene modules might play central roles in the transcriptional regulatory networks underlying cold acclimation in jojoba, in which the ICE1-CBF-SFR6 pathway activated stress response and plant defense, miR398-CSD1/2 and miR396-glutaredoxin regulated ROS homeostasis, and miR167-GDC1 modulated the protection of photosynthetic machinery. Our study will promote understanding of the molecular mechanism of cold acclimation and provide gene markers for the genetic breeding of jojoba for improving cold stress tolerance.

Integrated analysis of the transcriptome, sRNAome, and degradome reveals the network regulating fruit skin coloration in sponge gourd (Luffa cylindrica)

Sponge gourd fruit skin color is an important quality-related trait because it substantially influences consumer preferences. However, little is known about the miRNAs and genes regulating sponge gourd fruit skin coloration. This study involved an integrated analysis of the transcriptome, sRNAome, and degradome of sponge gourd fruit skins with green skin (GS) and white skin (WS). A total of 4,331 genes were differentially expressed between the GS and WS, with 2,442 down-regulated and 1,889 up-regulated genes in WS. The crucial genes involved in chlorophyll metabolism, chloroplast development, and chloroplast protection were identified (e.g., HEMA, CHLM, CRD1, POR, CAO, CLH, SGR, CAB, BEL1-like, KNAT, ARF, and peroxidase genes). Additionally, 167 differentially expressed miRNAs were identified, with 70 up-regulated and 97 down-regulated miRNAs in WS. Degradome sequencing identified 125 differentially expressed miRNAs and their 521 differentially expressed target genes. The miR156, miR159, miR166, miR167, miR172, and miR393 targeted the genes involved in chlorophyll metabolism, chloroplast development, and chloroplast protection. Moreover, a flavonoid biosynthesis regulatory network was established involving miR159, miR166, miR169, miR319, miR390, miR396, and their targets CHS, 4CL, bHLH, and MYB. The qRT-PCR data for the differentially expressed genes were generally consistent with the transcriptome results. Subcellular localization analysis of selected proteins revealed their locations in different cellular compartments, including nucleus, cytoplasm and endoplasmic reticulum. The study findings revealed the important miRNAs, their target genes, and the regulatory network controlling fruit skin coloration in sponge gourd.

Effects of scion-rootstock interaction on citrus fruit quality related to differentially expressed small RNAs

Rootstock affects the performance of the scion on productivity and fruit quality. Scions on trifoliate orange rootstock could produce better quality of fruits than on other commonly used rootstocks. This phenomenon has been well documented, but genetic factors affecting the fruit quality through interaction of scion and rootstock remained unclear. Fruits in different developmental stages from the Daya mandarin grafted on two genetically divergent rootstocks (Trifoliate orange (Poncirus trifoliata) (Z) and Hongju (Citrus reticulata) (H)) were used for fruit quality, expression of small RNA, and degradome sequencing analysis. Total soluble solids (TSS) of fruit on Z rootstock were significantly higher than those of on H. In contrast, titratable acid (TA) of fruit were lower on Z rootstock. One hundred and eight known and 145 novel microRNAs (miRNA), as well as 555 target genes were identified by in silico analysis and degradome sequencing. One hundred and twenty-nine differentially expressed miRNAs (DEMs) were identified by comparison of miRNA expression between the Daya mandarins grafted on two rootstocks in three different developmental stages. Correlation analysis with the fruits from three navel orange cultivars on two rootstocks showed that four DEMs (Cre-miR159-3p, Cre-miR160a, Cre-miR171b and Cre-miR399-3p) were significant correlated to TSS or TA in some of scion/rootstock combinations. Expressions of Cre-miR399-3p and its target gene INVE demonstrated consistent correlations with fruit quality of four scion varieties grated on Z rootstock, indicating that Cre-miR399–3p and its target gene INVE may be one of the key factors affecting fruit quality in Poncirus trifoliata rootstock.

Transcriptome and miRNAome analysis reveals components regulating tissue differentiation of bamboo shoots.

Primary thickening determines bamboo yield and wood property. However, little is known about the regulatory networks involved in this process. This study identified a total of 58,652 genes and 150 miRNAs via transcriptome and small RNA sequencing using the underground thickening shoot samples of wild-type (WT) Moso bamboo (Phyllostachys edulis) and a thick wall (TW) variant (P. edulis "Pachyloen") at five developmental stages (WTS1/TWS1-WTS5/TWS5). A total of 14,029 (65.17%) differentially expressed genes and 68 (45.33%) differentially expressed miRNAs were identified from the WT, TW, and WTTW groups. The first two groups were composed of four pairwise combinations, each between two successive stages (WTS2/TWS2_versus_WTS1/TWS1, WTS3/TWS3_versus_WTS2/TWS2, WTS4/TWS4_versus_WTS3/TWS3, and WTS5/TWS5_versus_WTS4/TWS4), and the WTTW group was composed of five combinations, each between two relative stages (TWS1-5_versus_WTS1-5). Additionally, among the phytohormones, zeatin showed more remarkable changes in concentrations than indole-3-acetic acid, gibberellic acid, and abscisic acid throughout the five stages in the WT and the TW groups. Moreover, 125 cleavage sites were identified for 387 miRNA-mRNA pairs via degradome sequencing (P < 0.05). The dual-luciferase reporter assay confirmed that 13 miRNAs bound to 12 targets. Fluorescence in situ hybridization localized miR166 and miR160 in the shoot apical meristem and the procambium of Moso bamboo shoots at the S1 stage. Thus, primary thickening is a complex process regulated by miRNA-gene-phytohormone networks, and the miRNAome and transcriptome dynamics regulate phenotypic plasticity. These findings provide insights into the molecular mechanisms underlying wood formation and properties and propose targets for bamboo breeding.

Organ-Specific Gene Expression Reveals the Role of the Cymbidium ensifolium-miR396/Growth-Regulating Factors Module in Flower Development of the Orchid Plant Cymbidium ensifolium.

Orchids are some of the most popular ornamental plants worldwide. Orchid floral morphology has increasingly attracted horticultural and commercial attention. Although multiple genes have been shown to be involved in the formation of the orchid flower, the underlying multi-level regulatory networks are largely unknown. In this study, we analyzed the ontogeny of flower development in Cymbidium ensifolium, a traditional orchid in the tropical and subtropical regions of Asia, by performing deep sequencing of the transcriptome of individual flower organs to discover organ-specific genes potentially involved in their growth. We identified 3,017 differentially-expressed genes (DEGs) during the development of various flower organs, and observed over-representation of GROWTH-REGULATING FACTORS (GRFs) specific to flower column (gynostemium). Eleven C. ensifolium GRFs (CeGRFs) from our transcriptome data clustered into five phylogenetic subgroups. Ten of these GRFs shared a region complementary to C. ensifolium microRNA396 (Ce-miR396), and degradome sequencing confirmed the cleavage of transcripts derived from seven CeGRFs. We cloned Ce-miR396 and used a protoplast-based transient expression system to overexpress it in Cymbidium protoplasts. We observed a significant decrease in the transcripts of several CeGRFs in flowers and leaves, indicating a potential role for miR396–GRF module in organ development through the cleavage of distinct CeGRFs. Temporal and spatial expression analysis indicated that most CeGRF transcripts accumulated in flower buds and column tissues, where Ce-miR396 expression was the lowest. Expression dynamics in wild type and floral-defective mutants further confirmed a strong correlation between Ce-miR396, CeGRFs, and flower organ development and column specification. Moreover, overexpression of Ce-miR396 in Nicotiana tabacum resulted in curved pistils and reduced fertility, implying that the conserved role of Ce-miR396 in floral development. These results provide tools to better understand the biological roles of GRFs in orchid development, and open new avenues for the diversification of orchid floral patterns.

An Integrated Bioinformatics and Functional Approach for miRNA Validation.

MicroRNAs (miRNAs) are small (20-24 nucleotides) non-coding ribo-regulatory molecules with significant roles in regulating target mRNA and long non-coding RNAs at transcriptional and post-transcriptional levels. Rapid advancement in the small RNA sequencing methods with integration of degradome sequencing has accelerated the understanding of miRNA-mediated regulatory hubs in plants and yielded extensive annotation of miRNAs and corresponding targets. However, it is becoming clear that large numbers of such annotations are questionable. Therefore, it is imperative to adopt reliable and strict bioinformatics pipelines for miRNA identification. Furthermore, sensitive methods are needed for validation and functional characterization of miRNA and its target(s). In this chapter, we have provided a comprehensive and streamlined methodology for miRNA identification and its functional validation in plants. This includes a combination of various in silico and experimental methodologies. To identify miRNA compendium from large-scale Next-Generation Sequencing (NGS) small RNA datasets, the miR-PREFeR (miRNA PREdiction From small RNA-Seq data) bioinformatics tool has been described. Also, a homology-based search protocol for finding members of a specific miRNA family has been discussed. The chapter also includes techniques to ascertain miRNA:target pair specificity using in silico target prediction from degradome NGS libraries using CleaveLand pipeline, miRNA:target validation by in planta transient assays, 5' RLM-RACE and expression analysis as well as functional techniques like miRNA overexpression, short tandem target mimic and resistant target approaches. The proposed strategy offers a reliable and sensitive way for miRNA:target identification and validation. Additionally, we strongly promulgate the use of multiple methodologies to validate a miRNA as well as its target.

Integration of Small RNA and Degradome Sequencing Reveals the Regulatory Network of Al-Induced Programmed Cell Death in Peanut.

Peanut is one of the most important oil crops in the world. In China, the peanut is highly produced in its southern part, in which the arable land is dominated by acid soil. At present, miRNAs have been identified in stress response, but their roles and mechanisms are not clear, and no miRNA studies have been found related to aluminum (Al)-induced programmed cell death (PCD). In the present study, transcriptomics, sRNAs, and degradome analysis in the root tips of two peanut cultivars ZH2 (Al-sensitive, S) and 99-1507 (Al-tolerant, T) were carried out. Here, we generated a comprehensive resource focused on identifying key regulatory miRNA-target circuits that regulate PCD under Al stress. Through deep sequencing, 2284 miRNAs were identified and 147 miRNAs were differentially expressed under Al stress. Furthermore, 19237 target genes of 749 miRNAs were validated by degradome sequencing. GO and KEGG analyses of differential miRNA targets showed that the pathways of synthesis and degradation of ketone bodies, citrate cycle (TCA cycle), and peroxisome were responded to Al stress. The combined analysis of the degradome data sets revealed 89 miRNA-mRNA interactions that may regulate PCD under Al stress. Ubiquitination may be involved in Al-induced PCD in peanut. The regulatory networks were constructed based on the differentially expressed miRNAs and their targets related to PCD. Our results will provide a useful platform to research on PCD induced by Al and new insights into the genetic engineering for plant stress response.

Integrated analysis of small RNA, transcriptome and degradome sequencing reveals that micro-RNAs regulate anther development in CMS cotton

Cytoplasmic male sterility (CMS) is an important phenotypic trait for heterosis utilization in cotton (Gosspium hirsutum L.). Micro-RNAs (miRNAs) play crucial roles in the pollen development of plants. To elucidate the roles of miRNA-target gene pairs in the CMS cotton line ganmian15A and its maintainer line ganmian15B, an integrated small RNA (sRNA), transcriptome, and degradome sequencing analysis of these lines at the pollen abortion stage was performed in the present research. A total of 339 known miRNAs and 961 conserved miRNAs were identified through high-throughput sequencing, among which 124 were differentially expressed between ganmian15A and ganmian15B. By comprehensive analysis of miRNA, transcriptome, and degradome sequencing, we identified 11 pairs of miRNAs/target genes at the pollen abortion stage. Among them, 9 miRNAs (4 novel and 5 known) were found for the first time to be associated with the occurrence of male sterility in cotton. The target genes of miRna-38 and miRna-23 were linked to cell wall development at the pollen stage. They may play an imperative role in CMS cotton by delaying the degradation of tapetal cells. Our findings revealed new insights into the genetic control of CMS occurrence, which will support the breeding of new sterile lines of cotton in the future.