Background: Small non-coding RNAs (sRNAs) play key roles in plant development, growth and responses to biotic and abiotic stresses. At least four classes of sRNAs have been well characterized in plants, including repeat-associated siRNAs (rasiRNAs), microRNAs (miRNAs), trans-acting siRNAs (tasiRNAs) and natural antisense transcript-derived siRNAs. Chinese fir (Cunninghamia lanceolata) is one of the most important coniferous evergreen tree species in China. No sRNA from Chinese fir has been described to date. Results: To obtain sRNAs in Chinese fir, we sequenced a sRNA library generated from seeds, seedlings, leaves, stems and calli, using Illumina high throughput sequencing technology. A comprehensive set of sRNAs were acquired, including conserved and novel miRNAs, rasiRNAs and tasiRNAs. With BLASTN and MIREAP we identified a total of 115 conserved miRNAs comprising 40 miRNA families and one novel miRNA with precursor sequence. The expressions of 16 conserved and one novel miRNAs and one tasiRNA were detected by RT-PCR. Utilizing real time RT-PCR, we revealed that four conserved and one novel miRNAs displayed developmental stage-specific expression patterns in Chinese fir. In addition, 209 unigenes were predicted to be targets of 30 Chinese fir miRNA families, of which five target genes were experimentally verified by 5' RACE, including a squamosa promoter-binding protein gene, a pentatricopeptide (PPR) repeat-containing protein gene, a BolA-like family protein gene, AGO1 and a gene of unknown function. We also demonstrated that the DCL3-dependent rasiRNA biogenesis pathway, which had been considered absent in conifers, existed in Chinese fir. Furthermore, the miR390-TAS3-ARF regulatory pathway was elucidated. Conclusions: We unveiled a complex population of sRNAs in Chinese fir through high throughput sequencing. This provides an insight into the composition and function of sRNAs in Chinese fir and sheds new light on land plant sRNA evolution.

Background: MicroRNAs (miRNAs) play key roles in diverse developmental processes, nutrient homeostasis and responses to biotic and abiotic stresses. The biogenesis and regulatory functions of miRNAs have been intensively studied in model angiosperms, such as Arabidopsis thaliana, Oryza sativa and Populus trichocarpa. However, global identification of Pinus densata miRNAs has not been reported in previous research. Results: Here, we report the identification of 34 conserved miRNAs belonging to 25 miRNA families from a P. densata mRNA transcriptome database using local BLAST and MIREAP programs. The primary and/or precursor sequences of 29 miRNAs were further confirmed by RT-PCR amplification and subsequent sequencing. The average value of the minimal folding free energy indexes of the 34 miRNA precursors was 0.92. Nineteen (58%) mature miRNAs began with a 5' terminal uridine residue. Analysis of miRNA precursors showed that 19 mature miRNAs were novel members of 14 conserved miRNA families, of which 17 miRNAs were further validated by subcloning and sequencing. Using real-time quantitative RT-PCR, we found that the expression levels of 7 miRNAs were more than 2-fold higher in needles than in stems. In addition, 72 P. densata mRNAs were predicted to be targets of 25 miRNA families. Four target genes, including a nodal modulator 1-like protein gene, two GRAS family transcription factor protein genes and one histone deacetylase gene, were experimentally verified to be the targets of 3 P. densata miRNAs, pde-miR162a, pde-miR171a and pde-miR482a, respectively. Conclusions: This study led to the discovery of 34 conserved miRNAs comprising 25 miRNA families from Pinus densata. These results lay a solid foundation for further studying the regulative roles of miRNAs in the development, growth and responses to environmental stresses in P. densata.

Drought stress is a serious problem worldwide that reduces crop productivity. The laser has been shown to play a positive physiological role in enhancing plant seedlings tolerance to various abiotic stresses. However, little information is available about the molecular mechanism of He-Ne laser irradiation induced physiological changes for wheat adapting to drought conditions. Here, we performed a large-scale transcriptome sequencing to determine the molecular roles of He-Ne laser pretreated wheat seedlings under drought stress. There were 98.822 transcripts identified, and, among them, 820 transcripts were found to be differentially expressed in He-Ne laser pretreated wheat seedlings under drought stress compared with drought stress alone. Furthermore, most representative transcripts related to photosynthesis, nutrient uptake and transport, homeostasis control of reactive oxygen species and transcriptional regulation were expressed predominantly in He-Ne laser pretreated wheat seedlings. Thus, the up-regulated physiological processes of photosynthesis, antioxidation and osmotic accumulation because of the modified expressions of the related genes could contribute to the enhanced drought tolerance induced by He-Ne laser pretreatment. These findings will expand our understanding of the complex molecular events associated with drought tolerance conferred by laser irradiation in wheat and provide abundant genetic resources for future studies on plant adaptability to environmental stresses.

Salt stress is a severe environmental factor that detrimentally affects wheat growth and production worldwide. Previous studies illustrate that exogenous jasmonic acid (JA) significantly improved salt tolerance in plants. However, little is known about the underlying molecular mechanisms of JA induced physiochemical changes in wheat seedlings under salt stress conditions. In this study, biophysiochemical and transcriptome analysis was conducted to explore the mechanisms of exogenous JA induced salt tolerance in wheat. Exogenous JA increased salt tolerance of wheat seedlings by alleviating membrane lipid oxidation, improving root morphology, enhancing the contents of ABA, JA and SA and increasing relative water content. In the RNA-seq profiles, we identified a total of 54,263 unigenes and 1,407 unigenes showed differentially expressed patterns in JA pretreated wheat seedlings exposed to salt stress comparing to those with salt stress alone. Subsequently, gene ontology (GO) and KEGG pathway enrichment analysis characterized that DEGs involved in linoleic acid metabolism and plant hormone signal transduction pathways were up-regulated predominantly in JA pretreated wheat seedlings exposed to salt stress. We noticed that genes that involved in antioxidative defense system and that encoding transcription factors were mainly up- or down-regulated. Moreover, SOD, POD, CAT and APX activities were increased in JA pretreated wheat seedlings exposed to salt stress, which is in accordance with the transcript profiles of the relevant genes. Taken together, our results demonstrate that the genes and enzymes involved in physiological and biochemical processes of antioxidant system, plant hormones and transcriptional regulation contributed to JA-mediated enhancement of salt tolerance in wheat. These findings will facilitate the elucidation of the potential molecular mechanisms associated with JA-dependent amelioration of salt stress in wheat and lay theoretical foundations for future studies concerning the improvement of plant tolerance to abiotic environmental stresses.