Evidence of Purifying Selection and Co-Evolution at the Fold-Back Arm of the Novel Precursor MicroRNA159 Gene in Phalaenopsis Species (Orchidaceae)

Background

MicroRNAs (miRNAs) are small, endogenously transcribed, non-protein-coding RNAs that play important roles in regulation of gene expression in animals and plants. Here, selective constraints on the novel precursor microRNA159 (pre-miR159) gene were investigated in 42 Phalaenopsis species (Orchidaceae).

Methods/Results

A novel precursor microRNA159 gene was isolated from 42 Phalaenopsis species using a new microRNA-PCR (miR-PCR) approach. Sequencing of pre-miR159 genes revealed differences from the canonical pre-miR159 gene in Phalaenopsis species and other plants. Results demonstrated that the 5′ and 3′ fold-back arms and the terminal loop of the novel pre-miR159 gene have undergone purifying selection and selective constraint for stabilizing the secondary hairpin structure. Two conserved motifs within the 5′ fold-back arm had the highest purifying selective pressure within the novel pre-miR159 gene. Evidence of sequence co-evolution between the 5′ and 3′ fold-back regions was observed.

Conclusions

Functional selective pressure might arise from the constraint of forming a hairpin structure and demonstrate co-evolution of sequences between the 5′ and 3′ fold-back regions of the novel pre-miR159 gene in Phalaenopsis species.     

Genome-Wide Identification of Reverse Complementary microRNA Genes in Plants

MicroRNAs (miRNAs) are ∼21-nucleotide small RNAs (sRNAs) with essential regulatory roles in plants. They are generated from stem-loop-structured precursors through two sequential Dicer-like 1 (DCL1)-mediated cleavages. To date, hundreds of plant miRNAs have been uncovered. However, the question, whether the sequences reverse complementary (RC) to the miRNA precursors could form hairpin-like structures and produce sRNA duplexes similar to the miRNA/miRNA* pairs has not been solved yet. Here, we interrogated this possibility in 16 plant species based on sRNA high-throughput sequencing data and secondary structure prediction. A total of 59 RC sequences with great potential to form stem-loop structures and generate miRNA/miRNA*-like duplexes were identified in ten plants, which were named as RC-miRNA precursors. Unlike the canonical miRNAs, only a few cleavage targets of the RC-miRNAs were identified in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), and none in Soybean (Glycine max) based on degradome data. Surprisingly, the genomic regions surrounding some of the RC-miRNA target recognition sites were observed to be specifically methylated in both Arabidopsis and rice. Taken together, we reported a new class of miRNAs, called RC-miRNAs, which were generated from the antisense strands of the miRNA precursors. Based on the results, we speculated that the mature RC-miRNAs might have subtle regulatory activity through target cleavages, but might possess short interfering RNA-like activity by guiding sequence-specific DNA methylation.     

OsABCG15 encodes a membrane protein that plays an important role in anther cuticle and pollen exine formation in rice

Key message

An ABC transporter gene ( OsABCG15 ) was proven to be involved in pollen development in rice. The corresponding protein was localized on the plasma membrane using subcellular localization.

Abstract

Wax, cutin, and sporopollenin are important for normal development of the anther cuticle and pollen exine, respectively. Their lipid soluble precursors, which are produced in the tapetum, are then secreted and transferred to the anther and microspore surface for polymerization. However, little is known about the mechanisms underlying the transport of these precursors. Here, we identified and characterized a member of the G subfamily of ATP-binding cassette (ABC) transporters, OsABCG15, which is required for the secretion of these lipid-soluble precursors in rice. Using map-based cloning, we found a spontaneous A-to-C transition in the fourth exon of OsABCG15 that caused an amino acid substitution of Thr-to-Pro in the predicted ATP-binding domain of the protein sequence. This osabcg15 mutant failed to produce any viable pollen and was completely male sterile. Histological analysis indicated that osabcg15 exhibited an undeveloped anther cuticle, enlarged middle layer, abnormal Ubisch body development, tapetum degeneration with a falling apart style, and collapsed pollen grains without detectable exine. OsABCG15 was expressed preferentially in the tapetum, and the fused GFP-OsABCG15 protein was localized to the plasma membrane. Our results suggested that OsABCG15 played an essential role in the formation of the rice anther cuticle and pollen exine. This role may include the secretion of the lipid precursors from the tapetum to facilitate the transfer of precursors to the surface of the anther epidermis as well as to microspores.     

Identification of Novel Oryza sativa miRNAs in Deep Sequencing-Based Small RNA Libraries of Rice Infected with Rice Stripe Virus

MicroRNAs (miRNAs) play essential regulatory roles in the development of eukaryotes. Methods based on deep-sequencing have provided a powerful high-throughput strategy for identifying novel miRNAs and have previously been used to identify over 100 novel miRNAs from rice. Most of these reports are related to studies of rice development, tissue differentiation, or abiotic stress, but novel rice miRNAs related to viral infection have rarely been identified. In previous work, we constructed and pyrosequenced the small RNA (sRNA) libraries of rice infected with Rice stripe virus and described the character of the small interfering RNAs (siRNA) derived from the RSV RNA genome. We now report the identification of novel miRNAs from the abundant sRNAs (with a minimum of 100 sequencing reads) in the sRNA library of RSV-infected rice. 7 putative novel miRNAs (pn-miRNAs) whose precursor sequences have not previously been described were identified and could be detected by Northern blot or RT-PCR, and were recognized as novel miRNAs (n-miRNAs). Further analysis showed that 5 of the 7 n-miRNAs were up-expressed while the other 2 n-miRNAs were down-expressed in RSV-infected rice. In addition, 23 pn-miRNAs that were newly produced from 19 known miRNA precursors were also identified. This is first report of novel rice miRNAs produced from new precursors related to RSV infection.     

Identification of four functionally important microRNA families with contrasting differential expression profiles between drought-tolerant and susceptible rice leaf at vegetative stage

Background

Developing drought-tolerant rice varieties with higher yield under water stressed conditions provides a viable solution to serious yield-reduction impact of drought. Understanding the molecular regulation of this polygenic trait is crucial for the eventual success of rice molecular breeding programmes. microRNAs have received tremendous attention recently due to its importance in negative regulation. In plants, apart from regulating developmental and physiological processes, microRNAs have also been associated with different biotic and abiotic stresses. Hence here we chose to analyze the differential expression profiles of microRNAs in three drought treated rice varieties: Vandana (drought-tolerant), Aday Sel (drought-tolerant) and IR64 (drought-susceptible) in greenhouse conditions via high-throughput sequencing.

Results

Twenty-six novel microRNA candidates involved in the regulation of diverse biological processes were identified based on the detection of miRNA*. Out of their 110 predicted targets, we confirmed 16 targets from 5 novel microRNA candidates. In the differential expression analysis, mature microRNA members from 49 families of known Oryza sativa microRNA were differentially expressed in leaf and stem respectively with over 28 families having at least a similar mature microRNA member commonly found to be differentially expressed between both tissues. Via the sequence profiling data of leaf samples, we identified osa-miR397a/b, osa-miR398b, osa-miR408-5p and osa-miR528-5p as being down-regulated in two drought-tolerant rice varieties and up-regulated in the drought-susceptible variety. These microRNAs are known to be involved in regulating starch metabolism, antioxidant defence, respiration and photosynthesis. A wide range of biological processes were found to be regulated by the target genes of all the identified differentially expressed microRNAs between both tissues, namely root development (5.3–5.7 %), cell transport (13.2–18.4 %), response to stress (10.5–11.3 %), lignin catabolic process (3.8–5.3 %), metabolic processes (32.1–39.5 %), oxidation-reduction process (9.4–13.2 %) and DNA replication (5.7–7.9 %). The predicted target genes of osa-miR166e-3p, osa-miR166h-5p*, osa-miR169r-3p* and osa-miR397a/b were found to be annotated to several of the aforementioned biological processes.

Conclusions

The experimental design of this study, which features rice varieties with different drought tolerance and tissue specificity (leaf and stem), has provided new microRNA profiling information. The potentially regulatory importance of the microRNA genes mentioned above and their target genes would require further functional analyses.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1851-3) contains supplementary material, which is available to authorized users.