Novel and mechanical stress-responsive MicroRNAs in Populus trichocarpa that are absent from Arabidopsis

MicroRNAs (miRNAs) are small, noncoding RNAs that can play crucial regulatory roles in eukaryotes by targeting mRNAs for silencing. To test whether miRNAs play roles in the regulation of wood development in tree species, we isolated small RNAs from the developing xylem of Populus trichocarpa stems and cloned 22 miRNAs. They are the founding members of 21 miRNA gene families for 48 miRNA sequences, represented by 98 loci in the Populus genome. A majority of these miRNAs were predicted to target developmental- and stress/defense-related genes and possible functions associated with the biosynthesis of cell wall metabolites. Of the 21 P. trichocarpa miRNA families, 11 have sequence conservation in Arabidopsis thaliana but exhibited species-specific developmental expression patterns, suggesting that even conserved miRNAs may have different regulatory roles in different species. Most unexpectedly, the remaining 10 miRNAs, for which 17 predicted targets were experimentally validated in vivo, are absent from the Arabidopsis genome, suggesting possible roles in tree-specific processes. In fact, the expression of a majority of the cloned miRNAs was upregulated or downregulated in woody stems in a manner consistent with tree-specific corrective growth against tension and compression stresses, two constant mechanical loads in trees. Our results show that plant miRNAs can be induced by mechanical stress and may function in one of the most critical defense systems for structural and mechanical fitness.     

植物microRNA的保守性及其分布的分析

MicroRNA(miRNA)是真核生物中具有重要调控作用的小分子非编码RNA。本文对miRNA官网miRBase数据库Release 22.1中隶属于植物界的绿藻门、苔藓植物门、蕨类植物门、裸子植物门、被子植物门共计82个物种的miRNA进行了统计分析。miRBase共收录植物miRNA 前体8 615个,成熟miRNA 10 414条,隶属于2 892个miRNA家族。绿藻门miRNA与其他4个门miRNA无同源性;对其他4个门植物miRNA的保守性进行研究,发现存在于2个植物门的miRNA家族有26个,属于中度保守miRNA家族;14个miRNA家族存在于3个及3个以上植物门中,属于高度保守miRNA家族,其中7个miRNA家族系苔藓、蕨类、裸子和被子植物共有,是植物中最保守的miRNA。分析表明,超过30个miRNA家族的植物有35种。进一步对40个中度或者高度保守miRNA在35种植物中的分布进行研究,发现miRNA家族及其成员在物种间的分布存在较大的差异。这些分布上的差异一方面反映不同植物中miRNA的研究深度不同,另一方面也反映出miRNA在植物进化过程中的适应性调整。研究不同植物中miRNA家族的分布,可在miRNA水平为植物早期进化同源性的研究提供分子依据。     

植物microRNA的保守性及其分布的分析

MicroRNA(miRNA)是真核生物中具有重要调控作用的小分子非编码RNA。本文对miRNA官网miRBase数据库Release 22.1中隶属于植物界的绿藻门、苔藓植物门、蕨类植物门、裸子植物门、被子植物门共计82个物种的miRNA进行了统计分析。miRBase共收录植物miRNA 前体8 615个,成熟miRNA 10 414条,隶属于2 892个miRNA家族。绿藻门miRNA与其他4个门miRNA无同源性;对其他4个门植物miRNA的保守性进行研究,发现存在于2个植物门的miRNA家族有26个,属于中度保守miRNA家族;14个miRNA家族存在于3个及3个以上植物门中,属于高度保守miRNA家族,其中7个miRNA家族系苔藓、蕨类、裸子和被子植物共有,是植物中最保守的miRNA。分析表明,超过30个miRNA家族的植物有35种。进一步对40个中度或者高度保守miRNA在35种植物中的分布进行研究,发现miRNA家族及其成员在物种间的分布存在较大的差异。这些分布上的差异一方面反映不同植物中miRNA的研究深度不同,另一方面也反映出miRNA在植物进化过程中的适应性调整。研究不同植物中miRNA家族的分布,可在miRNA水平为植物早期进化同源性的研究提供分子依据。     

Data mining for miRNAs and their targets in the Triticeae.

MicroRNAs (miRNAs) and the mRNA targets of miRNAs were identified by sequence complementarity within a DNA sequence database for species of the Triticeae. Data screening identified 28 miRNA precursor sequences from 15 miRNA families that contained conserved mature miRNA sequences within predicted stem-loop structures. In addition, the identification of 337 target sequences among Triticeae genes provided further evidence of the existence of 26 miRNA families in the cereals. MicroRNA targets included genes that are homologous to known targets in diverse model species as well as novel targets. MicroRNA precursors and targets were identified in 10 related species, though the great majority of them were identified in bread wheat, Triticum aestivum, and barley, Hordeum vulgare, the two species with the largest EST data sets among the Triticeae.     

Deep sequencing discovery and profiling of conserved and novel miRNAs in the ovule of <Emphasis Type="Italic">Ginkgo biloba</Emphasis>

Key message

High-throughput sequencing and subsequent analysis identified multiple miRNAs closely related to ovule, indicating that miRNAs are important in Ginkgo biloba ovule.

Abstract

MicroRNAs (miRNAs) are small, noncoding, regulatory RNAs that play crucial regulatory roles in the process of plant growth and development. However, limited information regarding their functions in gymnosperm reproduction is available. Here, we used high-throughput sequencing combined with computational analysis to identify and characterize miRNAs from ovules of G. biloba, and identified 34 conserved miRNA families and 99 novel miRNAs. The precursor sequences of several of the conserved and novel miRNAs were further validated by RT-PCR and sequencing. Furthermore, we found that some target genes, e.g. MYB, homeodomain-leucine zipper (HD-ZIPIII) and auxin response factor (ARF), may be involved in ovule development, and that the significantly enriched pathways of some miRNA targets were related to plant–pathogen interactions and the biosynthesis of secondary metabolites. Twenty-six conserved miRNA families were found to be expressed in both leaves and ovules, while miRNA156, miRNA164, miRNA167, miRNA169, miRNA172 and miRNA390 were up-regulated in ovules. Thus, multiple miRNAs closely related to G. biloba ovule development were identified, resulting in a greater understanding of the important regulatory functions of miRNAs in plant ovules.

     

Expression profiles of precursor and mature microRNAs under dehydration and high salinity shock in Populus euphratica

MicroRNAs (miRNAs) are small non-coding RNAs that play vital roles in plant abiotic stress responses via cleavage or translational inhibition of their target mRNAs. Populus euphratica is a typical stress-resistant sessile organism that grows in desert areas. Here, we identified sequences of 12 miRNA precursors from 11 families and 13 mature miRNAs from 12 families by PCR amplification in P. euphratica. To detect expression differences in mature miRNAs and their precursors under dehydration and high salinity shock in P. euphratica, we examined 14 miRNA precursors from 13 miRNA families and 17 mature miRNAs from 17 miRNA families using the SYBR Green RT–PCR assay. This is the first report of expression profiles for both precursor and mature miRNAs in P. euphratica. By profiling both the mature miRNAs and the precursors under abiotic stress shock, it was possible to identify miRNA whose processing is regulated during stress shock environments. A majority of the genes predicted to be targets for plant miRNAs are involved in development, stress resistance and metabolic processes. We have cloned and experimentally identified in vivo five of the predicted target genes and quantified the five target mRNAs from the same RNA sample simultaneously. Based on this study, we propose some regulatory pathways that illustrate the important role that miRNAs play in response to abiotic stress shock in P. euphratica.     

Identification of novel miRNAs and their target genes in <Emphasis Type="Italic">Eucalyptus grandis</Emphasis>

Despite Eucalyptus grandis being the most widely planted hardwood tree globally, along with the availability of a sequenced genome and easily accessible functional genetic tools, the quantities and roles of miRNA in its developmental processes remains largely unknown. In this study, we constructed small RNA libraries by high-throughput sequencing from Eucalyptus grandis samples, and 386 novel miRNAs were identified by miRDeep2. We found 179 novel miRNAs, 41 miRNA families, and 456 target genes in leaf samples, and 257 novel miRNAs, 61 miRNA families, and 483 target genes in stem samples. The function of the MIR396 family of miRNAs in Eucalyptus grandis was found to be mainly associated with the process of cell growth. By annotation analysis of miRNA targets, we found that some target genes, such as GRF, expansin-A15, and RPS2, had a close correlation in stem. Finally, the three randomly selected members of the MIR396 family were confirmed to express in Eucalyptus grandis by qRT-PCR, indicating that our reported miRNAs were existed. The identification of miRNAs and their target genes will lead to a greater understanding of the role of miRNAs in the physiology, growth, and development of Eucalyptus grandis trees.     

Identification and characterization of microRNAs and their target genes in Brassica oleracea

The microRNAs are a new class of small non-coding endogenous RNAs with lengths of approximately ~21 nt. MicroRNAs perform their biological function via the degradation of the target mRNAs or by inhibiting protein translation. Until recently, only limited numbers of miRNAs were identified in Brassica oleracea, a vegetable widely cultivated around the world. In present study, 193 potential miRNA candidates were identified from 17 expressed sequence tag (ESTs) and 152 genome survey sequences (GSSs) in B. oleracea. These miRNA candidates were classified into 70 families using a well-defined comparative genome-based computational analysis. Most miRNAs belong to the miRNA169, miR5021, miR156 and miR158 families. Of these, 36 miRNA families are firstly found in Brassica species. Around 1393 B. oleracea genes were predicted as candidate targets of 175 miRNAs. The mutual relationship between miRNAs and the candidate target genes was verified by checking differentially expression levels using quantitative real-time polymerase chain reaction (qRT-PCR) and 5' RLM-RACE analyses. These target genes participate in multiple biological and metabolic processes, including signal transduction, stress response, and plant development. Gene Ontology analysis shows that the 818, 514, and 265 target genes are involved in molecular functions, biological processes, and cellular component respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway enrichment analysis suggests that these miRNAs might regulate 186 metabolic pathways, including those of lipid, energy, starch and sucrose, fatty acid and nitrogen.     

Characterization of microRNAs Expressed during Secondary Wall Biosynthesis in Acacia mangium

MicroRNAs (miRNAs) play critical regulatory roles by acting as sequence specific guide during secondary wall formation in woody and non-woody species. Although thousands of plant miRNAs have been sequenced, there is no comprehensive view of miRNA mediated gene regulatory network to provide profound biological insights into the regulation of xylem development. Herein, we report the involvement of six highly conserved amg-miRNA families (amg-miR166, amg-miR172, amg-miR168, amg-miR159, amg-miR394, and amg-miR156) as the potential regulatory sequences of secondary cell wall biosynthesis. Within this highly conserved amg-miRNA family, only amg-miR166 exhibited strong differences in expression between phloem and xylem tissue. The functional characterization of amg-miR166 targets in various tissues revealed three groups of HD-ZIP III: ATHB8, ATHB15, and REVOLUTA which play pivotal roles in xylem development. Although these three groups vary in their functions, -psRNA target analysis indicated that miRNA target sequences of the nine different members of HD-ZIP III are always conserved. We found that precursor structures of amg-miR166 undergo exhaustive sequence variation even within members of the same family. Gene expression analysis showed three key lignin pathway genes: C4H, CAD, and CCoAOMT were upregulated in compression wood where a cascade of miRNAs was downregulated. This study offers a comprehensive analysis on the involvement of highly conserved miRNAs implicated in the secondary wall formation of woody plants.