Identification and Expression Analysis of microRNAs at the Grain Filling Stage in Rice(Oryza sativa L.)via Deep Sequencing

MicroRNAs (miRNAs) have been shown to play crucial roles in the regulation of plant development. In this study, high-throughput RNA-sequencing technology was used to identify novel miRNAs, and to reveal miRNAs expression patterns at different developmental stages during rice (Oryza sativa L.) grain filling. A total of 434 known miRNAs (380, 402, 390 and 392 at 5, 7, 12 and 17 days after fertilization, respectively.) were obtained from rice grain. The expression profiles of these identified miRNAs were analyzed and the results showed that 161 known miRNAs were differentially expressed during grain development, a high proportion of which were up-regulated from 5 to 7 days after fertilization. In addition, sixty novel miRNAs were identified, and five of these were further validated experimentally. Additional analysis showed that the predicted targets of the differentially expressed miRNAs may participate in signal transduction, carbohydrate and nitrogen metabolism, the response to stimuli and epigenetic regulation. In this study, differences were revealed in the composition and expression profiles of miRNAs among individual developmental stages during the rice grain filling process, and miRNA editing events were also observed, analyzed and validated during this process. The results provide novel insight into the dynamic profiles of miRNAs in developing rice grain and contribute to the understanding of the regulatory roles of miRNAs in grain filling.     

Characterization and Expression Patterns of microRNAs Involved in Rice Grain Filling

MicroRNAs (miRNAs) are upstream gene regulators of plant development and hormone homeostasis through their directed cleavage or translational repression of the target mRNAs, which may play crucial roles in rice grain filling and determining the final grain weight and yield. In this study, high-throughput sequencing was performed to survey the dynamic expressions of miRNAs and their corresponding target genes at five distinct developmental stages of grain filling. In total, 445 known miRNAs and 45 novel miRNAs were detected with most of them expressed in a developmental stage dependent manner, and the majority of known miRNAs, which increased gradually with rice grain filling, showed negatively related to the grain filling rate. Detailed expressional comparisons revealed a clear negative correlation between most miRNAs and their target genes. It was found that specific miRNA cohorts are expressed in a developmental stage dependent manner during grain filling and the known functions of these miRNAs are involved in plant hormone homeostasis and starch accumulation, indicating that the expression dynamics of these miRNAs might play key roles in regulating rice grain filling.     

Identification of miRNAs and Their Target Genes in Peach (Prunus persica L.) Using High-Throughput Sequencing and Degradome Analysis

MicroRNAs play critical roles in various biological and metabolic processes. The function of miRNAs has been widely studied in model plants such as Arabidopsis and rice. However, the number of identified miRNAs and related miRNA targets in peach (Prunus persica) is limited. To understand further the relationship between miRNAs and their target genes during tissue development in peach, a small RNA library and three degradome libraries were constructed from three tissues for deep sequencing. We identified 117 conserved miRNAs and 186 novel miRNA candidates in peach by deep sequencing and 19 conserved miRNAs and 13 novel miRNAs were further evaluated for their expression by RT-qPCR. The number of gene targets that were identified for 26 conserved miRNA families and 38 novel miRNA candidates, were 172 and 87, respectively. Some of the identified miRNA targets were abundantly represented as conserved miRNA targets in plant. However, some of them were first identified and showed important roles in peach development. Our study provides information concerning the regulatory network of miRNAs in peach and advances our understanding of miRNA functions during tissue development.     

Characterization and expression profiles of miRNAs in rice seeds

Small RNAs (sRNAs) are common and effective modulators of gene expression in eukaryotic organisms. To characterize the sRNAs expressed during rice seed development, massively parallel signature sequencing (MPSS) was performed, resulting in the obtainment of 797 399 22-nt sequence signatures, of which 111 161 are distinct ones. Analysis on the distributions of sRNAs on chromosomes showed that most sRNAs originate from interspersed repeats that mainly consist of transposable elements, suggesting the major function of sRNAs in rice seeds is transposon silencing. Through integrative analysis, 26 novel miRNAs and 12 miRNA candidates were identified. Further analysis on the expression profiles of the known and novel miRNAs through hybridizing the generated chips revealed that most miRNAs were expressed preferentially in one or two rice tissues. Detailed comparison of the expression patterns of miRNAs and corresponding target genes revealed the negative correlation between them, while few of them are positively correlated. In addition, differential accumulations of miRNAs and corresponding miRNA*s suggest the functions of miRNA*s other than being passenger strands of mature miRNAs, and in regulating the miRNA functions.     

Aluminum triggers broad changes in microRNA expression in rice roots

MicroRNAs are small 21-nucleotide RNA molecules with regulatory roles in development and in response to stress. Expression of some plant miRNAs has been specifically associated with responses to abiotic stresses caused by cold, light, iron, and copper ions. In acid soils, aluminum solubility increases, thereby causing severe damage to plants. Although physiological aspects of aluminum toxicity in plants have been well characterized, the molecular mediators are not fully elucidated. There have been no reports about miRNA responses to aluminum stress. Modulation of miRNA expression may constitute a key element to explain the mechanisms implicated in aluminum toxicity and tolerance. We examined the expression of at least one miRNA member from each miRNA family in rice roots of Oryza sativa spp indica cv. Embrapa Taim and Oryza sativa spp japonica cv. Nipponbare under high concentrations of aluminum. Forty-six miRNA families were effectively detected by quantitative PCR. Among these, 13 were down-regulated and six were up-regulated in roots of the Nipponbare cultivar after 8 h of aluminum treatment. In roots of the Embrapa Taim cultivar, five miRNAs were down-regulated and three were up-regulated. Analyses of their putative targets suggest that these rice miRNAs are involved in the regulation of various metabolic pathways in response to high concentrations of aluminum.     

The Regulatory Roles of microRNAs and Associated Target Genes during Early Somatic Embryogenesis in <i>Liriodendron Sino-Americanum</i>

Somatic cells respond to considerable stress, and go through a series of phytohormone pathways, then forming an embryo. The developmental process is recorded as somatic embryogenesis (SE). One of the key components regulating SE are the microRNAs (miRNAs). Despite previous studies, it is still not clear exactly how miRNAs exert their function of regulating targets during conditionally activated early SE. Here, we use Liriodendron sino-americanum as a model system and perform a combined analysis of microfluidic chips and degradome sequencing to study this process. We identified a total of 386 conserved miRNAs and 153 novel miRNAs during early SE. According to the ANOVA test, 239 miRNAs showed 12 distinct expression patterns. Through degradome sequencing, 419 targets and 198 targets were identified for 136 known miRNAs and 37 novel miRNAs, respectively. Gene Ontology (GO) and metabolism pathway enrichment analysis revealed that these targets were significantly involved in oxidation-reduction processes, calmodulin-mediated signal transduction pathways and carbohydrate metabolism. The genes that were related to stress responses, phytohormone pathways and plant metabolism were identified within the targets of miR319, miR395, miR408, miR472, miR482, miR390, miR2055, miR156, miR157, miR171, miR396, miR397, miR529, miR535 and miR159. According to promoter analysis, various cis-acting elements related to plant growth and development, phytohormones response and stress response were present in the promoter of the miRNAs. The differential expression patterns of 11 miRNA-target modules were confirmed by real-time quantitative PCR. The study demonstrated that the miRNA plays an important role in the early SE process by regulating its target and then participating in carbohydrate metabolism and stress response. It also provided a valuable resource for further research in determining the genetic mechanism of SE, and then facilitating breeding programs on plants.     

Small RNA and Degradome Sequencing Reveal Complex Roles of miRNAs and Their Targets in Developing Wheat Grains

Plant microRNAs (miRNAs) have been shown to play critical roles in plant development. In this study, we employed small RNA combined with degradome sequencing to survey development-related miRNAs and their validated targets during wheat grain development. A total of 186 known miRNAs and 37 novel miRNAs were identified in four small RNA libraries. Moreover, a miRNA-like long hairpin locus was first identified to produce 21~22-nt phased siRNAs that act in trans to cleave target mRNAs. A comparison of the miRNAomes revealed that 55 miRNA families were differentially expressed during the grain development. Predicted and validated targets of these development-related miRNAs are involved in different cellular responses and metabolic processes including cell proliferation, auxin signaling, nutrient metabolism and gene expression. This study provides insight into the complex roles of miRNAs and their targets in regulating wheat grain development.     

Expression patterns of microRNAs in different organs and developmental stages of a superhybrid rice LYP9 and its parental lines

Heterosis is an important phenomenon, and the molecular mechanisms underlying heterosis are still enigmatic. microRNAs (miRNAs) play vital roles in many aspects of plant development. A set of miRNAs was selected to investigate the roles of miRNAs in heterosis displayed in a superhybrid rice. We analysed the expression patterns of miRNAs in different organs and developmental stages of the superhybrid rice and its parental lines. All possible modes of miRNA action were observed, including additive, high‐ and low‐parent value, above high‐ and below low‐parent value. Different organs and developmental stages exhibited different modes of miRNA expression. Overall, the non‐additive mode is the predominant expression pattern of miRNAs observed in this superhybrid. Many heterotic QTL intervals harbour miRNAs, whose expression patterns reveal their specific roles in different organs and developmental stages. miRNAs regulate the expression levels of target genes that have important functions in plant development. The predominant non‐additive mode of miRNA expression pattern in the hybrid suggests that miRNAs play critical roles in hybrid development, in particular, those miRNAs located in the heterotic QTL intervals may have important roles in heterosis. Our research sheds new light on understanding of the molecular mechanisms of heterosis.     

Discovery and profiling of novel and conserved microRNAs during flower development in Carya cathayensis via deep sequencing

Hickory (Carya cathayensis Sarg.) is an economically important woody plant in China, but its long juvenile phase delays yield. MicroRNAs (miRNAs) are critical regulators of genes and important for normal plant development and physiology, including flower development. We used Solexa technology to sequence two small RNA libraries from two floral differentiation stages in hickory to identify miRNAs related to flower development. We identified 39 conserved miRNA sequences from 114 loci belonging to 23 families as well as two novel and ten potential novel miRNAs belonging to nine families. Moreover, 35 conserved miRNA*s and two novel miRNA*s were detected. Twenty miRNA sequences from 49 loci belonging to 11 families were differentially expressed; all were up-regulated at the later stage of flower development in hickory. Quantitative real-time PCR of 12 conserved miRNA sequences, five novel miRNA families, and two novel miRNA*s validated that all were expressed during hickory flower development, and the expression patterns were similar to those detected with Solexa sequencing. Finally, a total of 146 targets of the novel and conserved miRNAs were predicted. This study identified a diverse set of miRNAs that were closely related to hickory flower development and that could help in plant floral induction.     

Low genetic diversity and functional constraint of miRNA genes participating pollen–pistil interaction in rice

Key message

In this study, we sequenced and analyzed the expression and evolution of rice miRNA genesparticipating pollen-pistil interaction that is crucial to rice yield.

Abstract

Pollen–pistil interaction is an essential reproductive process for all flowering plants. While microRNAs (miRNAs) are important noncoding small RNAs that regulate mRNA levels in eukaryotic cells, there is little knowledge about which miRNAs involved in the early stages of pollen–pistil interaction in rice and how they evolve under this conserved process. In this study, we sequenced the small RNAs in rice from unpollinated pistil (R0), pistil from 5 min and 15 min after pollination, respectively, to identify known and novel miRNAs that are involved in this process. By comparing the corresponding mRNA-seq dataset, we identified a group of miRNAs with strong negative expression pattern with their target genes. Further investigation of all miRNA loci (MIRNAs) across 1083 public rice accessions revealed significantly reduced genetic diversity in MIRNAs with strong negative expression of their targets when comparing to those with little or no impact on targets during pollen–pistil interaction. Annotation of targets suggested that those MIRNAs with strong impact on targets were pronounced in cell wall related processes such as xylan metabolism. Additionally, plant conserved miRNAs, such as those with functions in gibberellic acid, auxin and nitrate signaling, were also with strong negative expression of their targets. Overall, our analyses identified key miRNAs participating pollen–pistil interaction and their evolutionary patterns in rice, which can facilitate the understanding of molecular mechanisms associated with seed setting.

     

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.