Identification of MicroRNAs and Transcript Targets in Camelina sativa by Deep Sequencing and Computational Methods

Camelina sativa is an annual oilseed crop that is under intensive development for renewable resources of biofuels and industrial oils. MicroRNAs, or miRNAs, are endogenously encoded small RNAs that play key roles in diverse plant biological processes. Here, we conducted deep sequencing on small RNA libraries prepared from camelina leaves, flower buds and two stages of developing seeds corresponding to initial and peak storage products accumulation. Computational analyses identified 207 known miRNAs belonging to 63 families, as well as 5 novel miRNAs. These miRNAs, especially members of the miRNA families, varied greatly in different tissues and developmental stages. The predicted miRNA target genes are involved in a broad range of physiological functions including lipid metabolism. This report is the first step toward elucidating roles of miRNAs in C. sativa and will provide additional tools to improve this oilseed crop for biofuels and biomaterials.     

Deep Sequencing and Microarray Hybridization Identify Conserved and Species-Specific MicroRNAs during Somatic Embryogenesis in Hybrid Yellow Poplar

Background

To date, several studies have indicated a major role for microRNAs (miRNAs) in regulating plant development, but miRNA-mediated regulation of the developing somatic embryo is poorly understood, especially during early stages of somatic embryogenesis in hardwood plants. In this study, Solexa sequencing and miRNA microfluidic chips were used to discover conserved and species-specific miRNAs during somatic embryogenesis of hybrid yellow poplar (Liriodendron tulipifera×L. chinense).

Methodology/Principal Findings

A total of 17,214,153 reads representing 7,421,623 distinct sequences were obtained from a short RNA library generated from small RNAs extracted from all stages of somatic embryos. Through a combination of deep sequencing and bioinformatic analyses, we discovered 83 sequences with perfect matches to known miRNAs from 33 conserved miRNA families and 273 species-specific candidate miRNAs. MicroRNA microarray results demonstrated that many conserved and species-specific miRNAs were expressed in hybrid yellow poplar embryos. In addition, the microarray also detected another 149 potential miRNAs, belonging to 29 conserved families, which were not discovered by deep sequencing analysis. The biological processes and molecular functions of the targets of these miRNAs were predicted by carrying out BLAST search against Arabidopsis thaliana GenBank sequences and then analyzing the results with Gene Ontology.

Conclusions

Solexa sequencing and microarray hybridization were used to discover 232 candidate conserved miRNAs from 61 miRNA families and 273 candidate species-specific miRNAs in hybrid yellow poplar. In these predicted miRNAs, 64 conserved miRNAs and 177 species-specific miRNAs were detected by both sequencing and microarray hybridization. Our results suggest that miRNAs have wide-ranging characteristics and important roles during all stages of somatic embryogenesis in this economically important species.     

Identification and profiling of conserved and novel microRNAs involved in oil and oleic acid production during embryogenesis in <Emphasis Type="Italic">Carya cathayensis</Emphasis> Sarg

MicroRNAs (miRNAs) are important regulators of plant development and fruit formation. Mature embryos of hickory (Carya cathayensis Sarg.) nuts contain more than 70% oil (comprising 90% unsaturated fatty acids), along with a substantial amount of oleic acid. To understand the roles of miRNAs involved in oil and oleic acid production during hickory embryogenesis, three small RNA libraries from different stages of embryogenesis were constructed. Deep sequencing of these three libraries identified 95 conserved miRNAs with 19 miRNA*s, 7 novel miRNAs (as well as their corresponding miRNA*s), and 26 potentially novel miRNAs. The analysis identified 15 miRNAs involved in oil and oleic acid production that are differentially expressed during embryogenesis in hickory. Among them, nine miRNA sequences, including eight conserved and one novel, were confirmed by qRT-PCR. In addition, 145 target genes of the novel miRNAs were predicted using a bioinformatic approach. Our results provide a framework for better understanding the roles of miRNAs during embryogenesis in hickory.     

Identification of MiRNA from Eggplant (Solanum melongena L.) by Small RNA Deep Sequencing and Their Response to Verticillium dahliae Infection

MiRNAs are a class of non-coding small RNAs that play important roles in the regulation of gene expression. Although plant miRNAs have been extensively studied in model systems, less is known in other plants with limited genome sequence data, including eggplant (Solanum melongena L.). To identify miRNAs in eggplant and their response to Verticillium dahliae infection, a fungal pathogen for which clear understanding of infection mechanisms and effective cure methods are currently lacking, we deep-sequenced two small RNA (sRNA) libraries prepared from mock-infected and infected seedlings of eggplants. Specifically, 30,830,792 reads produced 7,716,328 unique miRNAs representing 99 known miRNA families that have been identified in other plant species. Two novel putative miRNAs were predicted with eggplant ESTs. The potential targets of the identified known and novel miRNAs were also predicted based on sequence homology search. It was observed that the length distribution of obtained sRNAs and the expression of 6 miRNA families were obviously different between the two libraries. These results provide a framework for further analysis of miRNAs and their role in regulating plant response to fungal infection and Verticillium wilt in particular.     

Deep Sequencing of Maize Small RNAs Reveals a Diverse Set of MicroRNA in Dry and Imbibed Seeds

Seed germination plays a pivotal role during the life cycle of plants. As dry seeds imbibe water, the resumption of energy metabolism and cellular repair occur and miRNA-mediated gene expression regulation is involved in the reactivation events. This research was aimed at understanding the role of miRNA in the molecular control during seed imbibition process. Small RNA libraries constructed from dry and imbibed maize seed embryos were sequenced using the Illumina platform. Twenty-four conserved miRNA families were identified in both libraries. Sixteen of them showed significant expression differences between dry and imbibed seeds. Twelve miRNA families, miR156, miR159, miR164, miR166, miR167, miR168, miR169, miR172, miR319, miR393, miR394 and miR397, were significantly down-regulated; while four families, miR398, miR408, miR528 and miR529, were significantly up-regulated in imbibed seeds compared to that in dry seeds. Furthermore, putative novel maize miRNAs and their target genes were predicted. Target gene GO analysis was performed for novel miRNAs that were sequenced more than 50 times in the normalized libraries. The result showed that carbohydrate catabolic related genes were specifically enriched in the dry seed, while in imbibed seed target gene enrichment covered a broad range of functional categories including genes in amino acid biosynthesis, isomerase activity, ligase activity and others. The sequencing results were partially validated by quantitative RT-PCR for both conserved and novel miRNAs and the predicted target genes. Our data suggested that diverse and complex miRNAs are involved in the seed imbibition process. That miRNA are involved in plant hormone regulation may play important roles during the dry-imbibed seed transition.     

Analysis of expressed sequence tags from biodiesel plant Jatropha curcas embryos at different developmental stages

Jatropha curcas is considered a potential biodiesel feedstock plant whose seeds contain up to 40% oil. However, little is currently known about the seed biology of Jatropha. Therefore, it would be valuable to understand the mechanisms of development and lipid metabolism in Jatropha seeds. In the present study, three cDNA libraries were constructed with mRNA from Jatropha embryos at different stages of seed development. A total of 9844 expressed sequence tags (ESTs) were produced from these libraries, from which 1070 contigs and 3595 singletons were obtained. One hundred and seven unigenes were found to be differentially expressed in the three cDNA libraries of Jatropha embryos, indicating that these genes may play key roles in seed development. We have identified 59 and 61 unigenes that might be involved in the development and lipid metabolism in Jatropha seeds, respectively. Some of these genes may also play important roles in embryogenesis, morphogenesis, defense response and adaptive mechanisms in plants.     

Expression patterns of conserved microRNAs in the male gametophyte of loblolly pine (Pinus taeda)

MicroRNAs (miRNAs) are small RNAs that regulate genes involved in various aspects of plant development, but their presence and expression patterns in the male gametophytes of gymnosperms have not yet been established. Therefore, this study identified and compared the expression patterns of conserved miRNAs from two stages of the male gametophyte of loblolly pine (Pinus taeda), which are the mature (ungerminated) and germinated pollen. Microarray was used to identify conserved miRNAs that varied in expression between these two stages of the loblolly pine male gametophyte. Forty-seven conserved miRNAs showed significantly different expression levels between mature and germinated loblolly pine pollen. In particular, miRNAs representing 14 and 8 families were up- and down-regulated in germinated loblolly pine pollen, respectively. qRT-PCR was used to validate their expression patterns using representative miRNAs. Target genes and proteins were identified using psRNATarget program. Predicted targets of the 22 miRNA families belong mostly to classes of genes involved in defense/stress response, metabolism, regulation, and signaling. qRT-PCR was also used to validate the expression patterns of representative target genes. This study shows that conserved miRNAs are expressed in mature and germinated loblolly pine pollen. Many of these miRNAs are differentially expressed, which indicates that the two stages of the male gametophyte examined are regulated at the miRNA level. This study also expands our knowledge of the male gametophytes of seed plants by providing insights on some similarities and differences in the types and expression patterns of conserved miRNAs between loblolly pine with those of rice and Arabidopsis.