-
[show abstract]
[hide abstract]
ABSTRACT: MicroRNAs (miRNAs) play essential roles in numerous developmental and metabolic processes in animals and plants. Although the framework of miRNA biogenesis and function is established, the mechanism of miRNA degradation or modification remains to be investigated in plants. Mature miRNAs may be truncated or added nucleotides to generate variants. A detailed analysis of small RNA deep sequencing data sets resulted in the cloning of a large number of variants derived from larch miRNAs. Many 5'- and/or 3'-end truncated versions of miRNAs suggested that larch miRNAs might be degraded through either 5'-3' or 3'-5'. The relative abundance of variants truncated from 3'-end was higher than that of 5'-end for most miRNAs. The addition of adenine, uridine, and cytidine to the 3'-end of miRNAs was globally present, and the subtle variability in isomiR abundance might be regulated and biologically meaningful. It is the first report for cytidine addition in plant, and our examination of published small RNA deep sequencing data sets of Arabidopsis, rice, and moss suggests that cytidine addition to miRNA 3'-end exists broadly in plants. In addition, the nucleotide addition might be associated with 3'-5' miRNA degradation. Our results provide valuable information for a genome-wide survey of miRNA truncation and modification in larch or plants.
Planta 12/2012; · 3.00 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Small RNAs (sRNAs) are emerging as essential regulators of biological processes. However, several studies have reported that gymnosperms do not express appreciable amounts of 24-nt sRNAs, and conifers in particular may have a unique sRNA-silencing signature. Here, we compared the sRNA transcriptomes of Japanese larch somatic embryos (SE) and seedlings. SE sRNAs exhibited a length bias toward 24 nt, while seedlings showed a bias toward a 21-nt length. We also confirmed that larch is capable of producing 24-nt sRNAs based on a polyacrylamide gel analysis. The sRNA expression patterns varied according to developmental stage, which might be associated with Dicer-like 3 and RNA-dependent RNA polymerase2 (RDR2) levels. Our data suggest that many MIR loci that produce canonical microRNAs (miRNAs, 20-22 nt) and long sRNAs (23-26 nt) have dual functions; the latter were preferentially produced in SE compared to seedlings. However, the ratio of miRNAs to total sRNAs in seedlings was higher than in SE, and most miRNAs were upregulated in seedlings. Trans-acting small interfering RNAs (ta-siRNAs) generated from TAS3 triggered by miR390 were identified, and levels of the three detected ta-siRNAs peaked in mature embryos, which was not consistent with the lowest RDR6 level. These findings indicate that larch, and possibly other gymnosperms, shares a common sRNA pathway with other land plants, and that the sRNA distribution pattern varies according to developmental stage, which may be attributable to the expression of sRNA pathway genes.
Planta 09/2012; · 3.00 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Japanese larch (Larix leptolepis) is an ecologically and economically important species mainly grown in northeastern China, Japan and Europe. However, erratic flowering and poor germplasm resources caused by high embryo abortion rates have hampered breeding of Larix species. Somatic embryogenesis (SE) is an effective tool for the production of L. leptolepis with desirable characteristics, such as expression of totipotency, preparation of synthetic seeds, and genetic transformation. However, public genomic resources for this species are limited. We sequenced 591,759 raw expressed sequence tags (ESTs) from a 454 sequencing cDNA library of L. leptolepis somatic embryos, resulting in 572,403 high-quality reads. These reads were assembled into 70,927 unique sequences (UniGenes), including 32,321 contigs and 38,606 singletons. After removal of low-quality sequences, 65,115 UniGenes were annotated using the UniProtKB program. Based on their sequence similarity with known proteins, the matched 30,372 sequences from 664 species were estimated to represent approximately 19,000 unique genes. Gene ontology analysis revealed 21,324 UniGenes assigned to 51 categories. By Kyoto Encyclopedia of Genes and Genomes mapping, 25,773 transcripts were associated with 160 biochemical pathways. Further analysis screened four signal transduction pathways represented by 337 enzymes and 17 secondary metabolites. In silico analysis reveals that 207 UniESTs in Larix are homologous to MAPKs genes identified from other model plants, which may be involved in regulating SE development. This study provides an initial insight into the Larix transcriptomes of the pro-embryogenic mass and is a sound basis for future studies. KEY MESSAGE: We constructed a large, full-length 454 sequencing cDNA library of Larix leptolepis during somatic embryogenesis. More than 590,000 sequences were obtained and a deep-coverage EST database was constructed.
Plant Cell Reports 05/2012; 31(9):1637-57. · 2.27 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: MicroRNAs (miRNAs) are emerging as essential regulators of biological processes. Somatic embryogenesis is one of the most important techniques for gymnosperm-breeding programs, but there is little understanding of its underlying mechanism. To investigate the roles of miRNAs during somatic embryogenesis in larch, we constructed a small RNA library from somatic embryos. High-throughput sequencing of the library identified 83 conserved miRNAs from 35 families, 16 novel miRNAs, and 14 plausible miRNA candidates, with a high proportion specific to larch or gymnosperms. qRT-PCR analysis demonstrated that both the conserved and novel or candidate miRNAs were expressed in larch. Several miRNA precursor sequences were obtained via RACE. We predicted 110 target genes using bioinformatics, and validated 9 of them by 5' RACE. 11 conserved miRNA families including 17 miRNAs with critical functions in plant development and six target mRNAs were detected by qRT-PCR in the larch SE. Stage-specific expression of miRNAs and their targets indicate their possible modulation on SE of larch: miR171a/b might exert function on PEMs, while miR171c acts in the induction process of larch SE; miR397 and miR398 mainly involved in modulation of PEM propagation and transition to single embryo; miR162 and miR168 exert their regulatory function during total SE process, especially during stages 5-8; miR156, miR159, miR160, miR166, miR167, and miR390 might play regulatory roles during cotyledonary embryo development. These findings indicate that larch and possibly other gymnosperms have complex mechanisms of gene regulation involving specific and common miRNAs operating post-transcriptionally during embryogenesis.
Planta 04/2012; 236(2):647-57. · 3.00 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Somatic embryogenesis involves complex molecular signaling pathways. Deregulation of these signaling pathways can transform the embryogenic callus to non-embryogenic callus. To investigate the miRNA regulation underlying this detrimental transformation in Japanese Larch (Larix leptolepis), we compared miRNA expression profiles between embryogenic and non-embryogenic callus at day 3 and day 14 after sub-culture. Four miRNA families dominated the 165 differentially expressed miRNAs between embryogenic and non-embryogenic callus. Of the four, miR171 was up-regulated, and miR159, miR169, and miR172 were down-regulated in the embryogenic callus. These four families are all abiotic stress-induced miRNAs, and all target transcription factors that regulate a group of genes important for cell differentiation and development, including scarecrow-like (SCL) transcription factor (miR171), apetala2 (miR172), MYB transcription factors (miR159), and NF-YA transcription factor (miR169). Three down-regulated miRNA families in the embryogenic callus are also regulated by ABA, which further shed light into the potential mechanisms underlying the transformation of the embryogenic competence in L. leptolepis. This study represents the first report on the miRNA regulation of the embryogenic and non-embryogenic callus in plant, and thus these four miRNA families provide important clues for further functional investigation.
Biochemical and Biophysical Research Communications 07/2010; 398(3):355-60. · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Wood is the end product of secondary vascular system development, which begins from the cambium. The wood formation process includes four major stages: cell expansion, secondary wall biosynthesis, lignification, and programmed cell death. Transcriptional profiling is a rapid way to screen for genes involved in these stages and their transitions, providing the basis for understanding the molecular mechanisms that control this process.
In this study, cDNA microarrays were prepared from a subtracted cDNA library (cambium zone versus leaf) of Chinese white poplar (Populus tomentosa Carr.) and employed to analyze the transcriptional profiles during the regeneration of the secondary vascular system, a platform established in our previous study. Two hundred and seven genes showed transcript-level differences at the different regeneration stages. Dramatic transcriptional changes were observed at cambium initiation, cambium formation and differentiation, and xylem development, suggesting that these up- or downregulated genes play important roles in these stage transitions. Transcription factors such as AUX/IAA and PINHEAD, which were previously shown to be involved in meristem and vascular tissue differentiation, were strongly transcribed at the stages when cambial cells were initiated and underwent differentiation, whereas genes encoding MYB proteins and several small heat shock proteins were strongly transcribed at the stage when xylem development begins.
Employing this method, we observed dynamic changes in gene transcript levels at the key stages, including cambium initiation, cambium formation and differentiation, and xylem development, suggesting that these up- or downregulated genes are strongly involved in these stage transitions. Further studies of these genes could help elucidate their roles in wood formation.
BMC Genomics 02/2009; 10:215. · 4.07 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Large scale cDNA sequencing and genome tiling array studies have shown that around 50% of genomic DNA in humans is transcribed, of which 2% is translated into proteins and the remaining 98% is non-coding RNAs (ncRNAs). There is mounting evidence that these ncRNAs play critical roles in regulating DNA structure, RNA expression, protein translation and protein functions through multiple genetic mechanisms, and thus affect normal development of organisms at all levels. Today, we know very little about the regulatory mechanisms and functions of these ncRNAs, which is clearly essential knowledge for understanding the secret of life. To promote this emerging research subject of critical importance, in this paper we review (1) ncRNAs' past and present, (2) regulatory mechanisms and their functions, (3) experimental strategies for identifying novel ncRNAs, (4) experimental strategies for investigating their functions, and (5) methodologies and examples of the application of ncRNAs.
Science in China Series C Life Sciences 07/2006; 49(3):201-17. · 1.61 Impact Factor
