LaMIR166a-mediated auxin biosynthesis and signalling affect somatic embryogenesis in Larix leptolepis.

Somatic embryogenesis (SE) involves complex molecular signalling pathways. Understanding molecular mechanism of SE in Larix leptolepis (L. leptolepis) can aid research on genetic improvement of gymnosperms. Previously, we obtained five LaMIR166a (miR166a precursor) -overexpression embryonic cell lines in the gymnosperm Larix leptolepis. The proliferation rates of pro-embryogenic masses in transgenic and wild-type lines were calculated. Overexpression of the miR166a precursor LaMIR166a led to slower proliferation. When pro-embryogenic masses were transferred to maturation medium, the relative expression of LaMIR166a and miR166a in the LaMIR166a-overexpression lines was higher than in the wild-type during SE, while LaHDZ31-34 expression levels also increased without negative control by miR166, suggesting that regulation of HD-ZIP III by miR166a exits stage-specific characteristics. The key indole-3-acetic acid (IAA) biosynthetic gene Nitrilase of L. leptolepis (LaNIT) was identified and the effects of miR166a on auxin biosynthesis and signalling genes were studied. During SE, LaNIT, Auxin response factor1 (LaARF1) and LaARF2 mRNA levels and IAA contents were markedly higher in LaMIR166a-overexpression lines, which revealed lower deformity rate of embryos, indicating endogenous IAA synthesis is required for somatic embryo maturation in L. leptolepis. Additionally, the IAA biosynthesis and signalling genes showed similar expression patterns to LaHDZ31-34, suggesting HD-ZIP III genes have a positive regulatory effect on LaNIT. Our results suggest miR166a and LaHDZ31-34 have important roles in auxin biosynthesis and signalling during SE, which might determine if the somatic embryo normally developed to mature in L. leptolepis.

Whole genome sequencing and comparative transcriptome analysis of a novel seawater adapted, salt-resistant rice cultivar - sea rice 86.

BACKGROUND: Rice (Oryza sativa) is critical for human nutrition worldwide. Due to a growing population, cultivars that produce high yields in high salinity soil are of major importance. Here we describe the discovery and molecular characterization of a novel sea water adapted rice strain, Sea Rice 86 (SR86). RESULTS: SR86 can produce nutritious grains when grown in high salinity soil. Compared to a salt resistant rice cultivar, Yanfen 47 (YF47), SR86 grows in environments with up to 3X the salt content, and produces grains with significantly higher nutrient content in 12 measured components, including 2.9X calcium and 20X dietary fiber. Whole genome sequencing demonstrated that SR86 is a relatively ancient indica subspecies, phylogenetically close to the divergence point of the major rice varietals. SR86 has 12 chromosomes with a total genome size of 373,130,791 bps, slightly smaller than other sequenced rice genomes. Via comparison with 3000 rice genomes, we identified 42,359 putative unique, high impact variants in SR86. Transcriptome analysis of SR86 grown under normal and high saline conditions identified a large number of differentially expressed and salt-induced genes. Many of those genes fall into several gene families that have established or suggested roles in salt tolerance, while others represent potentially novel mediators of salt adaptation. CONCLUSIONS: Whole genome sequencing and transcriptome analysis of SR86 has laid a foundation for further molecular characterization of several desirable traits in this novel rice cultivar. A number of candidate genes related to salt adaptation identified in this study will be valuable for further functional investigation.

Transcriptome Analysis of mRNA and miRNA in Somatic Embryos of Larix leptolepis Subjected to Hydrogen Treatment.

Hydrogen is a therapeutic antioxidant that has been used extensively in clinical trials. It also acts as a bioactive molecule that can alleviate abiotic stress in plants. However, the biological effects of hydrogen in somatic embryos and the underlying molecular basis remain largely unknown. In this study, the morphological and physiological influence of exogenous H2 treatment during somatic embryogenesis was characterized in Larix leptolepis Gordon. The results showed that exposure to hydrogen increased the proportions of active pro-embryogenic cells and normal somatic embryos. We sequenced mRNA and microRNA (miRNA) libraries to identify global transcriptome changes at different time points during H2 treatment of larch pro-embryogenic masses (PEMs). A total of 45,393 mRNAs and 315 miRNAs were obtained. Among them, 4253 genes and 96 miRNAs were differentially expressed in the hydrogen-treated libraries compared with the control. Further, a large number of the differentially expressed mRNAs and miRNAs were related to reactive oxygen species (ROS) homeostasis and cell cycle regulation. We also identified 4399 potential target genes for 285 of the miRNAs. The differential expression data and the mRNA-miRNA interaction network described here provide new insights into the molecular mechanisms that determine the performance of PEMs exposed to H2 during 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.

Dynamic expression of small RNA populations in larch (Larix leptolepis).

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.

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

KEY MESSAGE: 142 miRNAs were identified and 38 miRNA targets were predicted, 4 of which were validated, in C. intermedia . The expression of 12 miRNAs in salt-stressed leaves was assessed by qRT-PCR. MicroRNAs (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.

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.

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

UNLABELLED: 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.

Examination of the Transcriptional Response to LaMIR166a Overexpression in Larix kaempferi (Lamb.) Carr.

The study of somatic embryogenesis can provide insight into early plant development. We previously obtained LaMIR166a-overexpressing embryonic cell lines of Larix kaempferi (Lamb.) Carr. To further elucidate the molecular mechanisms associated with miR166 in this species, the transcriptional profiles of wild-type (WT) and three LaMIR166a-overexpressing transgenic cell lines were subjected to RNA sequencing using the Illumina NovaSeq 6000 system. In total, 203,256 unigenes were generated using Trinity de novo assembly, and 2467 differentially expressed genes were obtained by comparing transgenic and WT lines. In addition, we analyzed the cleaved degree of LaMIR166a target genes LaHDZ31-34 in different transgenic cell lines by detecting the expression pattern of LaHdZ31-34, and their cleaved degree in transgenic cell lines was higher than that in WT. The downstream genes of LaHDZ31-34 were identified using Pearson correlation coefficients. Yeast one-hybrid and dual-luciferase report assays revealed that the transcription factors LaHDZ31-34 could bind to the promoters of LaPAP, LaPP1, LaZFP5, and LaPHO1. This is the first report of gene expression changes caused by LaMIR166a overexpression in Japanese larch. These findings lay a foundation for future studies on the regulatory mechanism of miR166.

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.

[Differentially expressed genes during Larix somatic embryomaturation and the expression profile of partial genes].

To study the molecular mechanism of Larix somatic embryogenesis, a differentially expressed cDNA library of Larix somatic embryo in the period of maturation was constructed using suppression subtractive hybridization (SSH). The cDNA from the cultures at the stage of somatic embryo maturation of embryogenic cell line Y35 of L. leptolepis xL. principis-rupprechtii was used as the tester and the cDNA from its subcultured callus was used as the driver. Eight hundreds randomly selected positive clones were sequenced, and 468 UniGenes were obtained finally. According to their function, these ESTs were classified into 19 categories and were involved in many biological process related to plant growth and development such as metabolism, transcription, signal transduction, transport facilitation, cell growth and division, cell structure, cell fate, protein synthesis or degradation, defense etc. Real-time PCR results of several ESTs showed that they were all differentially expressed at the different stages during cell line Y35 somatic embryo maturation.

Role of cin-miR2118 in drought stress responses in Caragana intermedia and Tobacco.

The miR2118 is highly conserved in leguminous plants. Its function is to regulate the expression of genes encoding the TIR-NBS-LRR resistance protein. In this study, cin-miR2118 from Caragana intermedia was functionally characterized, especially with regard to its role in drought stress resistance. Two target genes of cin-miR2118 were predicted and cloned, the occurrence of miR2118 target sequence in both genes indicated that they might be targets of cin-miR2118. We investigated the expression patterns of cin-miR2118 and its target genes in C. intermedia stems and found diverse changes in expression in response to drought stress. CiDR1 was negatively correlated with corresponding miR2118 expression while CiDR2 was positively correlated with cin-miR2118. For further study, induced tolerance was observed in the transgenic Tobacco with overexpression cin-miR2118 upon 140-min water deficiency. And the expression level of cin-miR2118 was dramatically increased under drought stress. These results reveal that cin-miR2118 exert positive effects on drought stress tolerance. In addition, our study unexpectedly found that overexpression of cin-miR2118 in Tobacco can cause phenotype changes, which suggested that cin-miR2118 may have a novel function as a growth regulator in Tobacco.

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.

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.

cDNA cloning, genomic organization and expression analysis during somatic embryogenesis of the translationally controlled tumor protein (TCTP) gene from Japanese larch (Larix leptolepis).

A full-length cDNA and genomic sequences of a translationally controlled tumor protein (TCTP) gene were isolated from Japanese larch (Larix leptolepis) and designated LaTCTP. The length of the cDNA was 1, 043 bp and contained a 504 bp open reading frame that encodes a predicted protein of 167 amino acids, characterized by two signature sequences of the TCTP protein family. Analysis of the LaTCTP gene structure indicated four introns and five exons, and it is the largest of all currently known TCTP genes in plants. The 5'-flanking promoter region of LaTCTP was cloned using an improved TAIL-PCR technique. In this region we identified many important potential cis-acting elements, such as a Box-W1 (fungal elicitor responsive element), a CAT-box (cis-acting regulatory element related to meristem expression), a CGTCA-motif (cis-acting regulatory element involved in MeJA-responsiveness), a GT1-motif (light responsive element), a Skn-1-motif (cis-acting regulatory element required for endosperm expression) and a TGA-element (auxin-responsive element), suggesting that expression of LaTCTP is highly regulated. Expression analysis demonstrated ubiquitous localization of LaTCTP mRNA in the roots, stems and needles, high mRNA levels in the embryonal-suspensor mass (ESM), browning embryogenic cultures and mature somatic embryos, and low levels of mRNA at day five during somatic embryogenesis. We suggest that LaTCTP might participate in the regulation of somatic embryo development. These results provide a theoretical basis for understanding the molecular regulatory mechanism of LaTCTP and lay the foundation for artificial regulation 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.