Genome-wide identification of Medicago truncatula microRNAs and their targets reveals their differential regulation by heavy metal.

We adopted a deep sequencing approach developed by Solexa (Illumina Inc., San Diego, CA, USA) to investigate global expression and complexity of microRNAs (miRNAs) and their targets from Medicago truncatula. Two small RNA libraries and two degradome libraries were constructed from mercury (Hg)-treated and Hg-free M. truncatula seedlings. For miRNAs, each library generated 18.5-18.6 million short sequences, resulting in 10.2-10.8 million clean reads. At least 52 new miRNA candidates with ≈ 21 nucleotides are perfectly matched to the M. truncatula genome. Statistical analysis on transcript abundance of the new candidate miRNAs revealed that most of them were differentially regulated by the heavy metal mercury Hg(II), with 12 miRNAs being specifically induced by Hg exposure. Additionally, we identified 201 individual miRNAs representing 63 known M. truncatula miRNA families, including 12 new conserved and one non-conserved miRNAs that have not been described before. Finally, 130 targets for 58 known (37 conserved and 21 non-conserved) miRNA families and 37 targets for 18 new M. truncatula-specific candidate miRNA families were identified by high-throughput degradome sequencing approach.

Transcriptome profiling of early developing cotton fiber by deep-sequencing reveals significantly differential expression of genes in a fuzzless/lintless mutant.

Cotton fiber as a single-celled trichome is a biological model system for studying cell differentiation and elongation. However, the complexity of its gene expression and regulatory mechanism allows only marginal progress. Here, we report the high-throughput tag-sequencing (Tag-seq) analysis using Solexa Genome Analyzer platform on transcriptome of -2 to 1 (fiber initiation, stage I) and 2-8 (fiber elongation, stage II) days post anthesis (DPA) cotton (Gossypium hirsutum) ovules (wild type: WT; Xuzhou 142 and its mutant: fuzzless/lintless or flM, in the same background). To this end, we sequenced 3.5-3.8 million tags representing 0.7-1.0 million unique transcripts for each library (WT1, WT2, M1, and M2). After removal of low quality tags, we obtained a total of 2,973,104, 3,139,306, 2,943,654, and 3,392,103 clean sequences that corresponded to 357,852, 280,787, 372,952, and 382,503 distinct tags for WT1, WT2, M1, and M2, respectively. All clean tags were aligned to the publicly available cotton transcript database (TIGR, http://www.tigr.org). About 15% of the distinct tags were uniquely mapped to the reference genes, and 31.4% of existing genes were matched by tags. The tag mapping to the database sequences generated 23,854, 24,442, 23,497, and 19,957 annotated genes for WT1, WT2, M1, and M2 libraries, respectively. Analyses of differentially expressed genes revealed the substantial changes in gene type and abundance between the wild type and mutant libraries. Among the 20 most differentially expressed genes in WT1/M1 and WT2/M2 libraries were cellulose synthase, phosphatase, and dehydrogenase, all of which are involved in the fiber cell development. Overall, the deep-sequencing analyses demonstrate the high degree of transcriptional complexity in early developing fibers and represent a major improvement over the microarrays for analyzing transcriptional changes on a large scale.

A cotton miRNA is involved in regulation of plant response to salt stress.

The present study functionally identified a new microRNA (microRNA ovual line 5, miRNVL5) with its target gene GhCHR from cotton (Gossypium hirsutum). The sequence of miRNVL5 precursor is 104 nt long, with a well developed secondary structure. GhCHR contains two DC1 and three PHD Cys/His-rich domains, suggesting that GhCHR encodes a zinc-finger domain-containing transcription factor. miRNVL5 and GhCHR express at various developmental stages of cotton. Under salt stress (50-400 mM NaCl), miRNVL5 expression was repressed, with concomitant high expression of GhCHR in cotton seedlings. Ectopic expression of GhCHR in Arabidopsis conferred salt stress tolerance by reducing Na(+) accumulation in plants and improving primary root growth and biomass. Interestingly, Arabidopsis constitutively expressing miRNVL5 showed hypersensitivity to salt stress. A GhCHR orthorlous gene At2g44380 from Arabidopsis that can be cleaved by miRNVL5 was identified by degradome sequencing, but no confidential miRNVL5 homologs in Arabidopsis have been identified. Microarray analysis of miRNVL5 transgenic Arabidopsis showed six downstream genes (CBF1, CBF2, CBF3, ERF4, AT3G22920, and AT3G49200), which were induced by salt stress in wild-type but repressed in miRNVL5-expressing Arabidopsis. These results indicate that miRNVL5 is involved in regulation of plant response to salt stress.

Analysis of phosphorus-deficient responsive miRNAs and cis-elements from soybean (Glycine max L.).

Phosphorus is one of the major factors controlling plant growth and productivity. Although physiological and molecular processes of P deficiency have been intensively investigated, our current understanding of the coordinated regulation of phosphate-responsive genes and signal networks is limited. In the present study, we performed a microarray-based genome-wide transcriptional analysis of miRNAs from soybean (Glycine max L.) under phosphate deficiency. miRNAs extracted from P-deficient and P-sufficient soybean were hybridized to an array containing 853 known plant miRNA sequences. An induction ratio significant at p<0.01 was observed for 57 miRNAs belonging to 27 families. Among these miRNA families, which differentially expressed, 7 and 8 were found to be up-regulated, whereas 17 and 6 were down-regulated in leaves and roots, respectively. Seven representative individual miRNAs were selected for qRT-PCR validation, and most showed an expression pattern similar to that on microarray. We further predicted P-responsive cis-elements from the promoters of miRNAs in response to and non-responding to P deficiency. In total, 125 putative cis-elements were identified for 24 soybean P-deficient responsive miRNAs. Interestingly, those miRNAs (54) not responding to P deficiency were also found to contain the same P-responsive motifs. A comparative analysis revealed that the frequency of the motif occurrence in the promoters of miRNA genes in response to P deficiency was higher than that of miRNA genes not responding to P deficiency. Our study provides initial evidence in soybean that a set of miRNAs with a high frequency of P-responsive cis-elements may coordinately regulate the plant response to P deficiency.