Xiaoxia Ma

MicroRNA-mediated signaling involved in plant root development.

MicroRNA (miRNA), recently recognized as a critical post-transcriptional modulator of gene expression, is involved in numerous biological processes in both animals and plants. Although eudicots and monocots, such as the model plants Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), possess distinct root systems, several homologous miRNA families are reported to be involved in root growth control in both plants. Consistent with recent notion that numerous signaling pathways are implicated in root development, these miRNAs are implicated in auxin signaling, nutrition metabolism, or stress response and have potential role in mediating the signal interactions. However, a recapitulative representation of these results is especially desired. This review provides a global view of the involvement of miRNAs in root development focusing on the two plants, Arabidopsis and rice. Based on current research advances, several innovative mechanisms of miRNA transcription, feedback regulatory circuit between miRNAs and transcription factors (TFs), and miRNA-mediated signal interactions are also discussed.

OsCAND1 Is Required for Crown Root Emergence in Rice

Crown roots are main components of the fibrous root system and important for crops to anchor and absorb water and nutrition. To understand the molecular mechanisms of crown root formation, we isolated a rice mutant defective in crown root emergence designated as Oscand1 (named after the Arabidopsis homologous gene AtCAND1). The defect of visible crown root in the Oscand1 mutant is the result of cessation of the G2/M cell cycle transition in the crown root meristem. Map-based cloning revealed that OsCAND1 is a homolog of Arabidopsis CAND1. During crown root primordium development, the expression of OsCAND1 is confined to the root cap after the establishment of fundamental organization. The transgenic plants harboring DR5::GUS showed that auxin signaling in crown root tip is abnormal in the mutant. Exogenous auxin application can partially rescue the defect of crown root development in Oscand1. Taken together, these data show that OsCAND1 is involved in auxin signaling to maintain the G2/M cell cycle transition in crown root meristem and, consequently, the emergence of crown root. Our findings provide new information about the molecular regulation of the emergence of crown root in rice.

Introns targeted by plant microRNAs: a possible novel mechanism of gene regulation

BackgroundIn plant cells, most microRNAs (miRNAs) perform cleavages of target mature mRNAs in the cytoplasm. A recent report of a miRNA pathway involved in DNA methylation in the rice nucleus raises the possibility that plant miRNAs could cleave intron-containing pre-mRNAs (the precursor of messenger RNAs) located in the nucleus.ResultsIn this study, we searched for the miRNA binding sites present within the introns of Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) genes. All miRNA—intron interactions predicted to result in cleavages were validated by using the public degradome sequencing data. As a result, 40 miRNA—intron pairs involving 25 miRNAs in Arabidopsis and 1912 pairs involving 91 miRNAs in rice were identified. For several rice genes, not all transcription forms (alternative splicing variants) were under similar regulation by specific miRNAs. Certain transcripts could escape cleavages due to the absence of intronic miRNA binding sites within these sequences. In some instances, specific cleaved intron remnants could be converted to double-stranded RNAs (dsRNAs) by RNA-dependent RNA polymerase 2. These dsRNAs could then be processed into 21- and 24-nt phased sRNAs by the activity of Dicer-like 1 and 3, respectively. The resultant siRNAs have the potential to be incorporated into Argonaute (AGO)-associated silencing complexes and result in cleavages of target pre-mRNA sequences.ConclusionsA regulatory model, miRNA—targeting of intron-containing pre-mRNAs—phased sRNAs—targeting of mature mRNAs is proposed, which further expands the potential modes of action of plant miRNAs.

Construction of small RNA-mediated gene regulatory networks in the roots of rice (Oryza sativa)

BackgroundThe root systems play essential roles for plants to anchorage to the soil, and to exploit the mineral and water resources. The molecular mechanisms underlying root development have been extensively studied to improve root system architecture, especially for the crops. Several microRNA (miRNA) families have been demonstrated to be involved in plant root development. However, whether the other small RNA (sRNA) species, which occupy a dominant portion of the plant endogenous sRNA population, possess potential roles in root development remains unclear.ResultsIn this study, by using sRNA high-throughput sequencing data, we made a comparison of the sRNA accumulation levels between the rice root tips and the whole roots. The sRNAs highly accumulated in the root tips and in the whole roots were extracted respectively. After Argonaute 1 (AGO1) enrichment analysis, the sRNAs with great potential of performing target cleavages were included for target prediction and degradome sequencing data-based validation. As a result, lists of the targets regulated by the AGO1-enriched sRNAs were obtained for both the root tips and the whole roots. Further evidences were identified from microarray data of the target genes to support some of the sRNA—target interactions. Specifically, the expression patterns of certain target genes in the root tips and the whole roots were contrary to those of the regulating sRNAs. Besides, several targets were indicated to play important roles in root development based on literature mining.ConclusionsTaken together, the regulatory networks mediated by the sRNAs highly accumulated in the root tips or in the whole roots could advance our current understanding of the sRNA-involved molecular mechanisms underlying rice root development. And, the sRNA—target lists could serve as the basis for further functional investigations.

A Rice Stromal Processing Peptidase Regulates Chloroplast and Root Development

The stromal processing peptidase (SPP) is a metalloendopeptidase that cleaves a broad range of precursor substrates. In this study, we isolated a rice mutant showing leaf chlorosis at the early seedling stage but inhibition of root growth during the whole growth period. Genetic analysis demonstrates that the phenotypes of the mutant were caused by a recessive single gene mutation. The mutated gene was cloned by map-based cloning, and was shown to encode an SPP. Sequence analysis showed a glutamate deletion in the highly conserved C-terminus of SPP in the mutant. The mutation of SPP in the mutant was verified by transgenic complementation. SPP is constitutively expressed in all tissues. Subcellular localization analysis indicates that SPP is targeted to the chloroplast. The expression of some genes associated with chloroplast development was decreased in young seedlings of the spp mutant, but not in 14-day-old seedlings. Western blot analysis revealed that the Rubisco small subunit is not precisely processed in the spp mutant in 7-day-old seedlings, but the processing activity in the spp mutant is restored in 14-day-old seedlings. Moreover, the expression levels of Cab1R and Cab2R for the light-harvesting chlorophyll a/b-binding protein (LHCP) were highly up-regulated in the transgenic plants with overexpression of SPP. The present results reveal that SPP is essential for chloroplast biogenesis at the early growth stage and for rice root development; this is the first report on the function of SPP in monocot plants.

Mechanisms of microRNA-mediated auxin signaling inferred from the rice mutant osaxr

Auxin, known as the central hormone, plays essential roles in plant growth and development. In auxin signaling pathways, the tiny RNA molecules, i.e. microRNAs (miRNAs), show their strong potential in modulating the auxin signal transduction. Recently, we isolated a novel auxin resistant rice mutant osaxr (Oryza sativa auxin resistant) that exhibited plethoric root defects. Microarray experiments were carried out to investigate the expression patterns of both the miRNAs and the protein-coding genes in osaxr. A number of miRNAs showed reduced auxin sensitivity in osaxr compared with the wild type (WT), which may contribute to the auxin-resistant phenotype of the mutant. Auxin response elements (AuxREs) were demonstrated to be more frequently present in the promoters of auxin-related miRNAs. In our previous report, a comparative analysis of miRNA and protein-coding gene expression datasets uncovered a number of reciprocally expressed miRNA–target pairs. A feedback circuit between miRNA and auxin response factor (ARF) was then proposed. Here, we will discuss in-depth some points raised in the previous report, in particular, the organ-specific expression patterns of miR164, the feedback regulatory model between miR167 and certain ARFs, and the potential signal interactions between auxin and nutrition or stress that are mediated by miRNAs in rice roots.

The use of high-throughput sequencing methods for plant microRNA research

MicroRNA (miRNA) acts as a critical regulator of gene expression at post-transcriptional and occasionally transcriptional levels in plants. Identification of reliable miRNA genes, monitoring the procedures of transcription, processing and maturation of the miRNAs, quantification of the accumulation levels of the miRNAs in specific biological samples, and validation of miRNA–target interactions become the basis for thoroughly understanding of the miRNA-mediated regulatory networks and the underlying mechanisms. Great progresses have been achieved for sequencing technology. Based on the high degree of sequencing depth and coverage, the high-throughput sequencing (HTS, also called next-generation sequencing) technology provides unprecedentedly efficient way for genome-wide or transcriptome-wide studies. In this review, we will introduce several HTS platform-based methods useful for plant miRNA research, including RNA-seq (RNA sequencing), RNA-PET-seq (paired end tag sequencing of RNAs), sRNA-seq (small RNA sequencing), dsRNA-seq (double-stranded RNA sequencing), ssRNA-seq (single-stranded RNA sequencing) and degradome-seq (degradome sequencing). In particular, we will provide some special cases to illustrate the novel use of HTS methods for investigation of the processing modes of the miRNA precursors, identification of the RNA editing sites on miRNA precursors, mature miRNAs and target transcripts, re-examination of the current miRNA registries, and discovery of novel miRNA species and novel miRNA–target interactions. Summarily, we opinioned that integrative use of the above mentioned HTS methods could make the studies on miRNAs more efficient.

Long non-coding RNAs: a novel endogenous source for the generation of Dicer-like 1-dependent small RNAs in Arabidopsis thaliana.

The biological relevance of long non-coding RNAs (lncRNAs) is emerging. Whether the lncRNAs could form structured precursors for small RNAs (sRNAs) production remains elusive. Here, 172 713 DCL1 (Dicer-like 1)-dependent sRNAs were identified in Arabidopsis. Except for the sRNAs mapped onto the microRNA precursors, the remaining ones led us to investigate their originations. Intriguingly, 65 006 sRNAs found their loci on 5891 lncRNAs. These sRNAs were sent to AGO (Argonaute) enrichment analysis. As a result, 1264 sRNAs were enriched in AGO1, which were then subjected to target prediction. Based on degradome sequencing data, 109 transcripts were validated to be targeted by 96 sRNAs. Besides, 44 lncRNAs were targeted by 23 sRNAs. To further support the origination of the DCL1-dependent sRNAs from lncRNAs, we searched for the degradome-based cleavage signals at either ends of the sRNA loci, which were supposed to be produced during DCL1-mediated processing of the long-stem structures. As a result, 63 612 loci were supported by degradome signatures. Among these loci, 6606 reside within the dsRNA-seq (double-stranded RNA sequencing) read-covered regions of 100 nt or longer. These regions were subjected to secondary structure prediction. And, 43 regions were identified to be capable of forming highly complementary long-stem structures. We proposed that these local long-stem structures could be recognized by DCL1 for cropping, thus serving as the sRNA precursors. We hope that our study could inspire more research efforts to study on the biological roles of the lncRNAs in plants.

Identification of the highly accumulated microRNA*s in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa)

Plant microRNAs (miRNAs) are crucial for the regulation of gene expression, which is involved in almost all the important biological processes. In the cytoplasm, the miRNA strand is selectively incorporated into a specific Argonaute (AGO)-associated gene silencing complex, while the miRNA* is degraded rapidly. Thus, most miRNA*s were thought to be biologically meaningless. Interestingly, several recent reports in both plants and animals have shaken this notion. Many miRNA*s were demonstrated to possess regulatory roles in gene expression. However, the low accumulation levels of most miRNA*s raise the question whether the activities of this small RNA (sRNA) species are widespread in plants. Here, by using publicly available sRNA high-throughput sequencing data, we found that the accumulation levels of several miRNA*s could be much higher than those of their miRNA partners in certain organs, mutants and/or AGO-associated silencing complexes of both Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). Based on target prediction and degradome sequencing data-based validation, some of these highly accumulated miRNA*s were indicated to possess cleavage-based potential regulatory role on certain targets. Besides, some interesting biological interpretations were obtained based on the accumulation patterns of the miRNA*s, the annotations of the target genes, and literature mining. Taken together, the expanded list of the highly accumulated miRNA*s along with their potential target genes discovered in this study further strengthened the current notion that certain members of the miRNA* species are biologically relevant, which needs further inspection.

Expression-Based Functional Investigation of the Organ-Specific MicroRNAs in Arabidopsis

MicroRNAs (miRNAs) play a pivotal role in plant development. The expression patterns of the miRNA genes significantly influence their regulatory activities. By utilizing small RNA (sRNA) high-throughput sequencing (HTS) data, the miRNA expression patterns were investigated in four organs (flowers, leaves, roots and seedlings) of Arabidopsis. Based on a set of criteria, dozens of organ-specific miRNAs were discovered. A dominant portion of the organ-specific miRNAs identified from the ARGONAUTE 4-enriched sRNA HTS libraries were highly expressed in flowers. Additionally, the expression of the precursors of the organ-specific miRNAs was analyzed. Degradome sequencing data-based approach was employed to identify the targets of the organ-specific miRNAs. The miRNA–target interactions were used for network construction. Subnetwork analysis unraveled some novel regulatory cascades, such as the feedback regulation mediated by miR161, the potential self-regulation of the genes miR172, miR396, miR398 and miR860, and the miR863-guided cleavage of the SERRATE transcript. Our bioinformatics survey expanded the organ-specific miRNA–target list in Arabidopsis, and could deepen the biological view of the miRNA expression and their regulatory roles.