Liwang Qi

Reference gene selection for quantitative real-time PCR normalization in Caragana intermedia under different abiotic stress conditions.

Quantitative real-time reverse transcription polymerase chain reaction (qPCR), a sensitive technique for gene expression analysis, depends on the stability of the reference genes used for data normalization. Caragana intermedia, a native desert shrub with strong drought-resistance, sand-fixing capacity and high forage value that is widespread in the desert land of west and northwest China, has not been investigated regarding the identification of reference genes suitable for the normalization of qPCR data. In this study, 10 candidate reference genes were analyzed in C. intermedia subjected to different abiotic (osmotic, salt, cold and heat) stresses, in two distinct plant organs (roots and leaves). The expression stability of these genes was assessed using geNorm, NormFinder and BestKeeper algorithms. The best-ranked reference genes differed across the different sets of samples, but UNK2, PP2A and SAND were the most stable across all tested samples. UNK2 and SAND would be appropriate for normalizing gene expression data for salt-treated roots, whereas the combination of UNK2, SAND and EF-1α would be appropriate for salt-treated leaves. UNK1, UNK2 and PP2A would be appropriate for PEG-treated (osmotic) roots, whereas the combination of TIP41 and PP2A was the most suitable for PEG-treated leaves. SAND, PP2A and TIP41 exhibited the most stable expression in heat-treated leaves. In cold-treated leaves, SAND and EF-1α were the most stably expressed. To further validate the suitability of the reference genes identified in this study, the expression levels of DREB1 and DREB2 (homologs of AtDREB1 and AtDREB2) were studied in parallel. This study is the first systematic analysis for the selection of superior reference genes for qPCR in C. intermedia under different abiotic stress conditions, and will benefit future studies on gene expression in C. intermedia and other species of the leguminous genus Caragana.

Genome-wide identification of microRNAs in larch and stage-specific modulation of 11 conserved microRNAs and their targets during somatic embryogenesis

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.

Four abiotic stress-induced miRNA families differentially regulated in the embryogenic and non-embryogenic callus tissues of Larix leptolepis.

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.

Changes in H2O2 content and antioxidant enzyme gene expression during the somatic embryogenesis of Larix leptolepis

Hydrogen peroxide (H2O2) content and transcript levels of genes encoding superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) antioxidant enzymes were investigated during different stages of somatic embryogenesis in Larix leptolepis. H2O2 content was lowest on day 0 when embryogenic callus was incubated on Murashige and Skoog (MS) medium supplemented with polyethylene glycol (PEG). This content began to increase when callus was transferred to the same medium but containing abscisic acid (ABA), and reached a peak at day 3 following incubation. The level of H2O2 dropped from day 7 to day 10, peaked at day 21, then dropped again at day 24 and day 35, but increased when somatic embryos reached maturity at day 45. Transcript levels of SOD, CAT, and APX were lowest when somatic embryos were cultured on callus induction medium without ABA. When calli were transferred onto somatic embryo maturation medium, expression patterns of SOD, CAT, and APX varied, and transcript levels at all stages were higher than those at d 0. SOD expression was highest at day 3. Whereas, CAT expression levels were low during early stages of somatic embryogenesis, but increased at day 21, declined at days 24 and 35, and then began to increase again at day 45. APX gene expression patterns were highest at days 3, 21, and 45. These results suggested that ABA was essential for promoting somatic embryogenesis of L. leptolepis. Moreover, ABA induced production of H2O2 and other active oxygen species (AOS), and mediated CAT, SOD, and APX gene expression in somatic embryogenesis of L. leptolepis.

Identification of microRNAs in Caragana intermedia by high-throughput sequencing and expression analysis of 12 microRNAs and their targets under salt stress

Key message142 miRNAs were identified and 38 miRNA targets were predicted, 4 of which were validated, inC. intermedia. The expression of 12 miRNAs in salt-stressed leaves was assessed by qRT-PCR.AbstractMicroRNAs (miRNAs) are endogenous small RNAs that play important roles in various biological and metabolic processes in plants. Caragana intermedia is an important ecological and economic tree species prominent in the desert environment of west and northwest China. To date, no investigation into C. intermedia miRNAs has been reported. In this study, high-throughput sequencing of small RNAs and analysis of transcriptome data were performed to identify both conserved and novel miRNAs, and also their target mRNA genes in C. intermedia. Based on sequence similarity and hairpin structure prediction, 132 putative conserved miRNAs (12 of which were confirmed to form hairpin precursors) belonging to 31 known miRNA families were identified. Ten novel miRNAs (including the miRNA* sequences of three novel miRNAs) were also discovered. Furthermore, 36 potential target genes of 17 known miRNA families and 2 potential target genes of 1 novel miRNA were predicted; 4 of these were validated by 5′ RACE. The expression of 12 miRNAs was validated in different tissues, and these and five target mRNAs were assessed by qRT-PCR after salt treatment. The expression levels of seven miRNAs (cin-miR157a, cin-miR159a, cin-miR165a, cin-miR167b, cin-miR172b, cin-miR390a and cin-miR396a) were upregulated, while cin-miR398a expression was downregulated after salt treatment. The targets of cin-miR157a, cin-miR165a, cin-miR172b and cin-miR396a were downregulated and showed an approximately negative correlation with their corresponding miRNAs under salt treatment. These results would help further understanding of miRNA regulation in response to abiotic stress in C. intermedia.

Transcriptome profiling and in silico analysis of somatic embryos in Japanese larch (Larix leptolepis)

AbstractJapanese 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.

Regulation of LaMYB33 by miR159 during maintenance of embryogenic potential and somatic embryo maturation in Larix kaempferi (Lamb.) Carr.

During the process of subculture of embryogenic cultures, sometimes they may become non-embryogenic, which is not desirable. However, this offers an opportunity to explore the mechanisms underlying cell fate determination and the maintenance of embryogenic potential of explants during the process of somatic embryogenesis. In a previous study, differential expression of microRNAs (miRNAs) has been detected between embryogenic and non-embryogenic cultures as well as during somatic embryo maturation of Larix kaempferi (Lamb.) Carr. However, little is known about the target genes of these miRNAs during these cellular differentiation processes. In this study, full-length cDNA of the MYB homologue from L. kaempferi, LaMYB33, was cloned. Sequence analysis showed that the miR159 target sequence is present in LaMYB33. The isolation of the miRNA-guided cleavage products of LaMYB33 further suggested that this gene is regulated by miRNA. LaMYB33 transcript levels between embryogenic and non-embryogenic cultures and during the late stage of somatic embryo maturation were measured and the results showed opposite patterns in the expression of LaMYB33 and mature miR159. Based on the relationships between the expression patterns of LaMYB33 and mature miR159, we concluded that the post-transcriptional regulation of LaMYB33 by miR159 participates in the maintenance of embryogenic or non-embryogenic potential and somatic embryo maturation, providing new insights into the regulatory mechanisms of somatic embryogenesis.

A genome-wide survey of microRNA truncation and 3' nucleotide addition events in larch (Larix leptolepis).

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.

Stage-specific regulation of four HD-ZIP III transcription factors during polar pattern formation in Larix leptolepis somatic embryos

Polar auxin transport provides a developmental signal for cell fate specification during somatic embryogenesis. Some members of the HD-ZIP III transcription factors participate in regulation of auxin transport, but little is known about this regulation in somatic embryogenesis. Here, four HD-ZIP III homologues from Larix leptolepis were identified and designated LaHDZ31, 32, 33 and 34. The occurrence of a miR165/166 target sequence in all four cDNA sequences indicated that they might be targets of miR165/166. Identification of the cleavage products of LaHDZ31 and LaHDZ32 in vivo confirmed that they were regulated by miRNA. Their mRNA accumulation patterns during somatic embryogenesis and the effects of 1-N-naphthylphthalamic acid (NPA) on their transcript levels and somatic embryo maturation were investigated. The results showed that the four genes had higher transcript levels at mature stages than at the proliferation stage, and that NPA treatment down-regulated the mRNA abundance of LaHDZ31, 32 and 33 at cotyledonary embryo stages, but had no effect on the mRNA abundance of LaHDZ34. We concluded that these four members of Larix HD-ZIP III family might participate in polar auxin transport and the development of somatic embryos, providing new insights into the regulatory mechanisms of somatic embryogenesis.

Deciphering Small Noncoding RNAs during the Transition from Dormant Embryo to Germinated Embryo in Larches (Larix leptolepis)

Small RNAs (sRNAs), as a key component of molecular biology, play essential roles in plant development, hormone signaling, and stress response. However, little is known about the relationships among sRNAs, hormone signaling, and dormancy regulation in gymnosperm embryos. To investigate the roles of sRNAs in embryo dormancy maintenance and release in Larix leptolepis, we deciphered the endogenous “sRNAome” in dormant and germinated embryos. High-throughput sequencing of sRNA libraries showed that dormant embryos exhibited a length bias toward 24-nt while germinated embryos showed a bias toward 21-nt lengths. This might be associated with distinct levels of RNA-dependent RNA polymerase2 (RDR2) and/or RDR6, which is regulated by hormones. Proportions of miRNAs to nonredundant and redundant sRNAs were higher in germinated embryos than in dormant embryos, while the ratio of unknown sRNAs was higher in dormant embryos than in germinated embryos. We identified a total of 160 conserved miRNAs from 38 families, 3 novel miRNAs, and 16 plausible miRNA candidates, of which many were upregulated in germinated embryos relative to dormant embryos. These findings indicate that larches and possibly other gymnosperms have complex mechanisms of gene regulation involving miRNAs and other sRNAs operating transcriptionally and posttranscriptionally during embryo dormancy and germination. We propose that abscisic acid modulates embryo dormancy and germination at least in part through regulation of the expression level of sRNA-biogenesis genes, thus changing the sRNA components.