Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis

MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are small noncoding RNAs that have recently emerged as important regulators of mRNA degradation, translational repression, and chromatin modification. In Arabidopsis thaliana, 43 miRNAs comprising 15 families have been reported thus far. In an attempt to identify novel and abiotic stress regulated miRNAs and siRNAs, we constructed a library of small RNAs from Arabidopsis seedlings exposed to dehydration, salinity, or cold stress or to the plant stress hormone abscisic acid. Sequencing of the library and subsequent analysis revealed 26 new miRNAs from 34 loci, forming 15 new families. Two of the new miRNAs from three loci are members of previously reported miR171 and miR319 families. Some of the miRNAs are preferentially expressed in specific tissues, and several are either upregulated or downregulated by abiotic stresses. Ten of the miRNAs are highly conserved in other plant species. Fifty-one potential targets with diverse function were predicted for the newly identified miRNAs based on sequence complementarity. In addition to miRNAs, we identified 102 other novel endogenous small RNAs in Arabidopsis. These findings suggest that a large number of miRNAs and other small regulatory RNAs are encoded by the Arabidopsis genome and that some of them may play important roles in plant responses to environmental stresses as well as in development and genome maintenance.     

Heavy metal-regulated new microRNAs from rice

MicroRNAs (miRNAs) are a novel class of short, endogenous non-coding small RNAs that have the ability to base pair with their target mRNAs to repress their translation or induce their degradation in both plants and animals. To identify heavy metal stress-regulated novel miRNAs, we constructed a library of small RNAs from rice seedlings that were exposed to toxic levels of cadmium (Cd²⁺). Sequencing of the library and subsequent analysis revealed 19 new miRNAs representing six families. These cloned new rice miRNAs have sequence conservation neither in Arabidopsis nor in any other species. Most of the new rice miRNAs were up- or down-regulated in response to the metal exposure. On the base of sequence complementarity, a total of 34 miRNA targets were predicted, of which 23 targets are functionally annotated and the other 11 records belong to unknown proteins. Some predicted targets of miRNAs are associated with the regulation of the response to heavy metal-induced stresses. In addition to the new miRNAs, we detected nine previously reported miRNAs and 56 other novel endogenous small RNAs in rice. These findings suggest that the number of new miRNAs in rice is unsaturated and some of them may play critical roles in plant responses to environmental stresses.     

Common functions for diverse small RNAs of land plants

Endogenous small RNAs, including microRNAs (miRNAs) and short interfering RNAs (siRNAs), are critical components of plant gene regulation. Some abundant miRNAs involved in developmental control are conserved between anciently diverged plants, while many other less-abundant miRNAs appear to have recently emerged in the Arabidopsis thaliana lineage. Using large-scale sequencing of small RNAs, we extended the known diversity of miRNAs in basal plants to include 88 confidently annotated miRNA families in the moss Physcomitrella patens and 44 in the lycopod Selaginella moellendorffii. Cleavage of 29 targets directed by 14 distinct P. patens miRNA families and a trans-acting siRNA (ta-siRNA) was experimentally confirmed. Despite a core set of 12 miRNA families also expressed in angiosperms, weakly expressed and apparently lineage-specific miRNAs accounted for the majority of miRNA diversity in both species. Nevertheless, the molecular functions of several of these lineage-specific small RNAs matched those of angiosperms, despite dissimilarities in the small RNA sequences themselves, including small RNAs that mediated negative feedback regulation of the miRNA pathway and miR390-dependent ta-siRNAs that guided the cleavage of AUXIN RESPONSE FACTOR mRNAs. Diverse, lineage-specific, small RNAs can therefore perform common biological functions in plants.     

A transcriptome‐wide study on the microRNA‐ and the Argonaute 1‐enriched small RNA‐mediated regulatory networks involved in plant leaf senescence

Leaf senescence is an important physiological process during the plant life cycle. However, systemic studies on the impact of microRNAs (miRNAs) on the expression of senescence‐associated genes (SAGs) are lacking. Besides, whether other Argonaute 1 (AGO1)‐enriched small RNAs (sRNAs) play regulatory roles in leaf senescence remains unclear. In this study, a total of 5,123 and 1,399 AGO1‐enriched sRNAs, excluding miRNAs, were identified in Arabidopsis thaliana and rice (Oryza sativa), respectively. After retrieving SAGs from the Leaf Senescence Database, all of the AGO1‐enriched sRNAs and the miRBase‐registered miRNAs of these two plants were included for target identification. Supported by degradome signatures, 200 regulatory pairs involving 120 AGO1‐enriched sRNAs and 40 SAGs, and 266 regulatory pairs involving 64 miRNAs and 42 SAGs were discovered in Arabidopsis. Moreover, 13 genes predicted to interact with some of the above‐identified target genes at protein level were validated as regulated by 17 AGO1‐enriched sRNAs and ten miRNAs in Arabidopsis. In rice, only one SAG was targeted by three AGO1‐enriched sRNAs, and one SAG was targeted by miR395. However, five AGO1‐enriched sRNAs were conserved between Arabidopsis and rice. Target genes conserved between the two plants were identified for three of the above five sRNAs, pointing to the conserved roles of these regulatory pairs in leaf senescence or other developmental procedures. Novel targets were discovered for three of the five AGO1‐enriched sRNAs in rice, indicating species‐specific functions of these sRNA–target pairs. These results could advance our understanding of the sRNA‐involved molecular processes modulating leaf senescence.     

Cloning and characterization of miRNAs from maize seedling roots under low phosphorus stress

MicroRNAs (miRNAs) are a class of small, non-coding regulatory RNAs that regulate gene expression by guiding target mRNA cleavage or translational inhibition in plants and animals. In this study, a small RNA library was constructed to identify conserved miRNAs as well as novel miRNAs in maize seedling roots under low level phosphorus stress. Twelve miRNAs were identified by high throughput sequencing of the library and subsequent analysis, two belong to conserved miRNA families (miRNA399b and miRNA156), and the remaining ten are novel and one of latter is conserved in gramineous species. Based on sequence homology, we predicted 125 potential target genes of these miRNAs and then expression patterns of 7 miRNAs were validated by semi-RT-PCR analysis. MiRNA399b, Zma-miR3, and their target genes (Zmpt1 and Zmpt2) were analyzed by real-time PCR. It is shown that both miRNA399b and Zma-miR3 are induced by low phosphorus stress and regulated by their target genes (Zmpt1 and Zmpt2). Moreover, Zma-miR3, regulated by two maize inorganic phosphate transporters as a newly identified miRNAs, would likely be directly involved in phosphate homeostasis, so was miRNA399b in Arabidopsis and rice. These results indicate that both conserved and maize-specific miRNAs play important roles in stress responses and other physiological processes correlated with phosphate starvation, regulated by their target genes. Identification of these differentially expressed miRNAs will facilitate us to uncover the molecular mechanisms underlying the progression of maize seedling roots development under low level phosphorus stress.     

Conservation and divergence in plant microRNAs

MicroRNAs (miRNAs) are a class of small, non-coding RNAs that regulate gene expression in eukaryotic cells. The past decade has seen an explosion in our understanding of the sets of miRNA genes encoded in the genomes in different species of plants and the mechanisms by which miRNAs interact with target RNAs. A subset of miRNA families (and their binding sites in target RNAs) are conserved between angiosperms and basal plants, suggesting they predate the divergence of existing lineages of plants. However, the majority of miRNA families expressed by any given plant species have a narrow phylogenetic distribution. As a group, these "young" miRNAs genes appear to be evolutionarily fluid and lack clearly understood biological function. The goal of this review is to summarize our understanding of the sets of miRNA genes and miRNA targets that exist in various plant species and to discuss hypotheses that explain the patterns of conservation and divergence observed among microRNAs in plants.     

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.     

Duplication and expression analysis of multicopy miRNA gene family members in Arabidopsis and rice

To understand the expansion ofmulticopy microRNA （miRNA） families in plants, we localized the reported miRNA genes from Arabidopsis and rice to their chromosomes, respectively, and observed that 37% of 117 miRNA genes from Arabidopsis and 35% of 173 miRNA genes from rice were segmental duplications in the genome. In order to characterize whether the expression diversification has occurred among plant multicopy miRNA family members, we designed PCR primers targeting 48 predicted miRNA precursors from 10 families in Arabidopsis and rice. Results from RT-PCR data suggest that the transcribed precursors of members within the same miRNA family were present at different expression levels. In addition, although miRl60 and miR162 sequences were conserved in Arabidopsis and rice, we found that the expression patterns of these genes differed between the two species. These data suggested that expression diversification has occurred in multicopy miRNA families, increasing our understanding of the expression regulation of miRNAs in plants.