MicroRNAs from Holarrhena pubescens stems: Identification by small RNA Sequencing and their Potential Contribution to Human Gene Targets.

Holarrhena pubescens is an effective medicinal plant from the Apocynaceae family, widely distributed over the Indian subcontinent and extensively used by Ayurveda and ethno-medicine systems without apparent side effects. We postulated that miRNAs, endogenous non-coding small RNAs that regulate gene expression at the post-transcriptional level, may, after ingestion into the human body, contribute to the medicinal properties of plants of this species by inducing regulated human gene expression to modulate. However, knowledge is scarce about miRNA in Holarrhena. In addition, to test the hypothesis on the potential pharmacological properties of miRNA, we performed a high-throughput sequencing analysis using the Next Generation Sequencing Illumina platform; 42,755,236 raw reads have been generated from H. pubescens stems from a library of small RNA isolated, identifying 687 known and 50 new miRNAs led. The novel H. pubescens miRNAs were predicted to regulate specific human genes, and subsequent annotations of gene functions suggested a possible role in various biological processes and signaling pathways, such as Wnt, MAPK, PI3K-Akt, and AMPK signaling pathways and endocytosis. The association of these putative targets with many diseases, including cancer, congenital malformations, nervous system disorders, and cystic fibrosis, has been demonstrated. The top hub proteins STAT3, MDM2, GSK3B, NANOG, IGF1, PRKCA, SNAP25, SRSF1, HTT, and SNCA show their interaction with human diseases, including cancer and cystic fibrosis. To our knowledge, this is the first report of uncovering H. pubescens miRNAs based on high-throughput sequencing and bioinformatics analysis. This study has provided new insight into a potential cross-species control of human gene expression. The potential for miRNA transfer should be evaluated as one possible mechanism of action to account for the beneficial properties of this valuable species.

Screening of differentially expressed microRNAs and target genes in two potato varieties under nitrogen stress.

BACKGROUND: A reasonable supply of nitrogen (N) fertilizer is essential for obtaining high-quality, high-level, and stable potato yields, and an improvement in the N utilization efficiency can effectively reduce N fertilizer use. It is important to use accurate, straightforward, and efficient transgenic breeding techniques for the identification of genes that can improve nitrogen use efficiency, thus enabling us to achieve the ultimate goal of breeding N-efficient potato varieties. In recent years, some of the mechanisms of miRNAs have been elucidated via the analysis of the correlation between the expression levels of potato miRNA target genes and regulated genes under conditions of stress, but the role of miRNAs in the inhibition/expression of key genes regulating N metabolism under N stress is still unclear. Our study aimed to identify the role played by specific enzymes and miRNAs in the responses of plants to N stress. RESULTS: The roots and leaves of the N-efficient potato variety, Yanshu4 ("Y"), and N-inefficient potato variety, Atlantic ("D"), were collected at the seedling and budding stages after they were exposed to different N fertilizer treatments. The miRNAs expressed differentially under the two types of N stress and their corresponding target genes were first predicted using miRNA and degradome analysis. Then, quantitative polymerase chain reaction (qRT-PCR) was performed to verify the expression of differential miRNAs that were closely related to N metabolism. Finally, the shearing relationship between stu-miR396-5p and its target gene StNiR was determined by analyzing luciferase activity levels. The results showed that NiR activity increased significantly with an increase in the applied N levels from the seedling stage to the budding stage, and NiR responded significantly to different N treatments. miRNA sequencing enabled us to predict 48 families with conserved miRNAs that were mainly involved in N metabolism, carbon metabolism, and amino acid biosynthesis. The differences in the expression of the following miRNAs were identified via screening (high expression levels and P < 0.05): stu-miR396-5p, stu-miR408b-3p_R-1, stu-miR3627-3p, stu-miR482a-3p, stu-miR8036-3p, stu-miR482a-5p, stu-miR827-5p, stu-miR156a_L-1, stu-miR827-3p, stu-miR172b-5p, stu-miR6022-p3_7, stu-miR398a-5p, and stu-miR166c-5p_L-3. Degradome analysis showed that most miRNAs had many-to-many relationships with target genes. The main target genes involved in N metabolism were NiR, NiR1, NRT2.5, and NRT2.7. qRT-PCR analysis showed that there were significant differences in the expression levels of stu-miR396-5p, stu-miR8036-3p, and stu-miR482a-3p in the leaves and roots of the Yanshu4 and Atlantic varieties at the seedling and budding stages under conditions that involved no N and excessive N application; the expression of these miRNAs was induced in response to N stress. The correlation between the differential expression of stu-miR396-5p and its corresponding target gene NiR was further verified by determining the luciferase activity level and was found to be strongly negative. CONCLUSION: The activity of NiR was significantly positively correlated with N application from the seedling to the budding stage. Differential miRNAs and target genes showed a many-to-many relationship with each other. The expression of stu-miR396-5p, stu-miR482a-3p, and stu-miR8036-3p in the roots and leaves of the Yanshu4 and Atlantic varieties at the seedling and budding stages was notably different under two types of N stress. Under two types of N stress, stu-miR396-5p was down-regulated in Yanshu4 in the seedling-stage and shoot-stage roots, and up-regulated in seedling-stage roots and shoot-stage leaves; stu-miR482a-3p was up-regulated in the seedling and shoot stages. The expression of stu-miR8036-3p was up-regulated in the leaves and roots at the seedling and budding stages, and down-regulated in roots under both types of N stress. The gene expressing the key enzyme involved in N metabolism, StNiR, and the stu-miR396-5p luciferase assay reporter gene had a strong regulatory relationship with each other. This study provides candidate miRNAs related to nitrogen metabolism and highlights that differential miRNAs play a key role in nitrogen stress in potato, providing insights for future research on miRNAs and their target genes in nitrogen metabolic pathways and breeding nitrogen-efficient potatoes.

Gametophyte genome activation occurs at pollen mitosis I in maize.

Flowering plants alternate between multicellular haploid (gametophyte) and diploid (sporophyte) generations. Pollen actively transcribes its haploid genome, providing phenotypic diversity even among pollen grains from a single plant. In this study, we used allele-specific RNA sequencing of single pollen precursors to follow the shift to haploid expression in maize pollen. We observed widespread biallelic expression for 11 days after meiosis, indicating that transcripts synthesized by the diploid sporophyte persist long into the haploid phase. Subsequently, there was a rapid and global conversion to monoallelic expression at pollen mitosis I, driven by active new transcription from the haploid genome. Genes showed evidence of increased purifying selection if they were expressed after (but not before) pollen mitosis I. This work establishes the timing during which haploid selection may act in pollen.

Silencing of StRIK in potato suggests a role in periderm related to RNA processing and stress.

BACKGROUND: The periderm is a protective barrier crucial for land plant survival, but little is known about genetic factors involved in its development and regulation. Using a transcriptomic approach in the cork oak (Q. suber) periderm, we previously identified an RS2-INTERACTING KH PROTEIN (RIK) homologue of unknown function containing a K homology (KH)-domain RNA-binding protein, as a regulatory candidate gene in the periderm. RESULTS: To gain insight into the function of RIK in the periderm, potato (S. tuberosum) tuber periderm was used as a model: the full-length coding sequence of RIK, hereafter referred to as StRIK, was isolated, the transcript profile analyzed and gene silencing in potato performed to analyze the silencing effects on periderm anatomy and transcriptome. The StRIK transcript accumulated in all vegetative tissues studied, including periderm and other suberized tissues such as root and also in wounded tissues. Downregulation of StRIK in potato by RNA interference (StRIK-RNAi) did not show any obvious effects on tuber periderm anatomy but, unlike Wild type, transgenic plants flowered. Global transcript profiling of the StRIK-RNAi periderm did show altered expression of genes associated with RNA metabolism, stress and signaling, mirroring the biological processes found enriched within the in silico co-expression network of the Arabidopsis orthologue. CONCLUSIONS: The ubiquitous expression of StRIK transcript, the flower associated phenotype and the differential expression of StRIK-RNAi periderm point out to a general regulatory role of StRIK in diverse plant developmental processes. The transcriptome analysis suggests that StRIK might play roles in RNA maturation and stress response in the periderm.

Different Patterns of mRNA Nuclear Retention during Meiotic Prophase in Larch Microsporocytes.

Recent studies show a crucial role of post-transcriptional processes in the regulation of gene expression. Our research has shown that mRNA retention in the nucleus plays a significant role in such regulation. We studied larch microsporocytes during meiotic prophase, characterized by pulsatile transcriptional activity. After each pulse, the transcriptional activity is silenced, but the transcripts synthesized at this time are not exported immediately to the cytoplasm but are retained in the cell nucleus and especially in Cajal bodies, where non-fully-spliced transcripts with retained introns are accumulated. Analysis of the transcriptome of these cells and detailed analysis of the nuclear retention and transport dynamics of several mRNAs revealed two main patterns of nuclear accumulation and transport. The majority of studied transcripts followed the first one, consisting of a more extended retention period and slow release to the cytoplasm. We have shown this in detail for the pre-mRNA and mRNA encoding RNA pol II subunit 10. In this pre-mRNA, a second (retained) intron is posttranscriptionally spliced at a precisely defined time. Fully mature mRNA is then released into the cytoplasm, where the RNA pol II complexes are produced. These proteins are necessary for transcription in the next pulse to occur.mRNAs encoding translation factors and SERRATE followed the second pattern, in which the retention period was shorter and transcripts were rapidly transferred to the cytoplasm. The presence of such a mechanism in various cell types from a diverse range of organisms suggests that it is an evolutionarily conserved mechanism of gene regulation.

Ubiquitin-dependent Argonauteprotein MEL1 degradation is essential for rice sporogenesis and phasiRNA target regulation.

MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1), a rice (Oryza sativa) Argonaute (AGO) protein, has been reported to function specifically at premeiotic and meiotic stages of germ cell development and is associated with a novel class of germ cell-specific small noncoding RNAs called phased small RNAs (phasiRNAs). MEL1 accumulation is temporally and spatially regulated and is eliminated after meiosis. However, the metabolism and turnover (i.e. the homeostasis) of MEL1 during germ cell development remains unknown. Here, we show that MEL1 is ubiquitinated and subsequently degraded via the proteasome pathway in vivo during late sporogenesis. Abnormal accumulation of MEL1 after meiosis leads to a semi-sterile phenotype. We identified a monocot-specific E3 ligase, XBOS36, a CULLIN RING-box protein, that is responsible for the degradation of MEL1. Ubiquitination at four K residues at the N terminus of MEL1 by XBOS36 induces its degradation. Importantly, inhibition of MEL1 degradation either by XBOS36 knockdown or by MEL1 overexpression prevents the formation of pollen at the microspore stage. Further mechanistic analysis showed that disrupting MEL1 homeostasis in germ cells leads to off-target cleavage of phasiRNA target genes. Our findings thus provide insight into the communication between a monocot-specific E3 ligase and an AGO protein during plant reproductive development.

Selenium and anthocyanins share the same transcription factors R2R3MYB and bHLH in wheat.

Anthocyanins and selenium have vital biological functions for human and plants, they were investigated thoroughly and separately in plants. Previous studies indicated pigmented fruits and vegetables had higher selenium concentration, but whether there is a relationship between anthocyanins and selenium is unclear. In this study, a combined phenotypic and genotypic methodological approach was undertaken to explore the potential relationship between anthocyanins and selenium accumulation by using phenotypic investigation and RNA-seq analysis. The results showed that pigmented cultivars enrichment in Se is a general phenomenon observed for these tested species, this due to pigmented cultivars have higher Se efficiency absorption. Se flow direction mainly improve concentration of S-rich proteins of LMW-GS. This may be a result of the MYB and bHLH co-regulate anthocyanins biosynthesis and Se metabolism at the transcriptional level. This thesis addresses a neglected aspect of the relevant relationship between anthocyanins and selenium.

Relationship between Secondary Metabolism and miRNA for Important Flavor Compounds in Different Tissues of Tea Plant (Camellia sinensis) As Revealed by Genome-Wide miRNA Analysis.

This study investigated the regulatory relationship between important flavor compounds and microRNA (miRNA) in nine different tissues of tea plant by analyzing the related metabolites, small RNAs (sRNAs), degradome, and coexpression network. A total of 272 differential expressed miRNAs (DEmiRNAs) were obtained, including 198 conserved miRNAs and 74 novel miRNAs. Meanwhile, the expression patterns of miR159-GAMYB, miR167-ARF, and miR396-GRF pairs were investigated by quantitative real-time polymerase chain reaction (qRT-PCR) and the target sites were verified by 5'RNA ligase-mediated RACE (5' RLM-RACE). Further coexpression analysis showed that the content of gallated catechins was significantly and negatively correlated with the expression of miR156, but positively correlated with the expression of miR166 and miR172. Additionally, the expression of miR169a, miR169l, and miR319h was shown to be positively correlated with the content of nongallated catechins and the experssion levels of ANRa, ANRb, and LARb. Moreover, important volatile compounds, such as linalool, geraniol, and 2-phenylethanol, were found to be highly positively correlated with the expression of miR171o, miRN71a, miRN71b, miRN71c, and miRN71d. Our data indicate that these miRNAs may play important roles in regulating the biosynthesis of flavor compounds in different tissues of tea plant.

Transcriptional, metabolic, physiological and developmental responses of switchgrass to phosphorus limitation.

Knowing how switchgrass (Panicum virgatum L.) responds and adapts to phosphorus (P)-limitation will aid efforts to optimize P acquisition and use in this species for sustainable biomass production. This integrative study investigated the impacts of mild, moderate, and severe P-stress on genome transcription and whole-plant metabolism, physiology and development in switchgrass. P-limitation reduced overall plant growth, increased root/shoot ratio, increased root branching at moderate P-stress, and decreased root diameter with increased density and length of root hairs at severe P-stress. RNA-seq analysis revealed thousands of genes that were differentially expressed under moderate and severe P-stress in roots and/or shoots compared to P-replete plants, with many stress-induced genes involved in transcriptional and other forms of regulation, primary and secondary metabolism, transport, and other processes involved in P-acquisition and homeostasis. Amongst the latter were multiple miRNA399 genes and putative targets of these. Metabolite profiling showed that levels of most sugars and sugar alcohols decreased with increasing P stress, while organic and amino acids increased under mild and moderate P-stress in shoots and roots, although this trend reversed under severe P-stress, especially in shoots.

Potato CYCLING DOF FACTOR 1 and its lncRNA counterpart StFLORE link tuber development and drought response.

Plants regulate their reproductive cycles under the influence of environmental cues, such as day length, temperature and water availability. In Solanum tuberosum (potato), vegetative reproduction via tuberization is known to be regulated by photoperiod, in a very similar way to flowering. The central clock output transcription factor CYCLING DOF FACTOR 1 (StCDF1) was shown to regulate tuberization. We now show that StCDF1, together with a long non-coding RNA (lncRNA) counterpart, named StFLORE, also regulates water loss through affecting stomatal growth and diurnal opening. Both natural and CRISPR-Cas9 mutations in the StFLORE transcript produce plants with increased sensitivity to water-limiting conditions. Conversely, elevated expression of StFLORE, both by the overexpression of StFLORE or by the downregulation of StCDF1, results in an increased tolerance to drought through reducing water loss. Although StFLORE appears to act as a natural antisense transcript, it is in turn regulated by the StCDF1 transcription factor. We further show that StCDF1 is a non-redundant regulator of tuberization that affects the expression of two other members of the potato StCDF gene family, as well as StCO genes, through binding to a canonical sequence motif. Taken together, we demonstrate that the StCDF1-StFLORE locus is important for vegetative reproduction and water homeostasis, both of which are important traits for potato plant breeding.

Reproductive phasiRNAs regulate reprogramming of gene expression and meiotic progression in rice.

Plant spermatogenesis is a complex process that directly affects crop breeding. A rapid change in gene abundance occurs at early meiosis prophase, when gene regulation is selective. However, how these genes are regulated remains unknown. Here, we show that rice reproductive phasiRNAs are essential for the elimination of a specific set of RNAs during meiotic prophase I. These phasiRNAs cleave target mRNAs in a regulatory manner such that one phasiRNA can target more than one gene, and/or a single gene can be targeted by more than one phasiRNA to efficiently silence target genes. Our investigation of phasiRNA-knockdown and PHAS-edited transgenic plants demonstrates that phasiRNAs and their nucleotide variations are required for meiosis progression and fertility. This study highlights the importance of reproductive phasiRNAs for the reprogramming of gene expression during meiotic progression and establishes a basis for future studies on the roles of phasiRNAs with a goal of crop improvement.

Comparative transcriptome analyses in contrasting onion (Allium cepa L.) genotypes for drought stress.

Onion (Allium cepa L.) is an important vegetable crop widely grown for diverse culinary and nutraceutical properties. Being a shallow-rooted plant, it is prone to drought. In the present study, transcriptome sequencing of drought-tolerant (1656) and drought-sensitive (1627) onion genotypes was performed to elucidate the molecular basis of differential response to drought stress. A total of 123206 and 139252 transcripts (average transcript length: 690 bases) were generated after assembly for 1656 and 1627, respectively. Differential gene expression analyses revealed upregulation and downregulation of 1189 and 1180 genes, respectively, in 1656, whereas in 1627, upregulation and downregulation of 872 and 1292 genes, respectively, was observed. Genes encoding transcription factors, cytochrome P450, membrane transporters, and flavonoids, and those related to carbohydrate metabolism were found to exhibit a differential expression behavior in the tolerant and susceptible genotypes. The information generated can facilitate a better understanding of molecular mechanisms underlying drought response in onion.

Comparative analysis and functional identification of temperature-sensitive miRNA in Arabidopsis anthers.

The anther is one of the most vulnerable organs to temperature stress. Many previous works focused on the genes regulating anthers development, but few results of miRNA in anther development were reported. In order to investigate the transcriptional regulation of temperature-sensitive anther development, RNA-Sequencing was used to study micRNA in anthers of Arabidopsis thaliana under 16 °C and 27 °C. A total of 46.26 million clean reads were generated and mapped to 715,748 small RNA sequences containing 281 miRNAs. Then 13 differentially expressed (DE) miRNAs, containing 3 novel miRNAs were found. Comprehensive analysis of miRNA expression showed 7 miRNAs were down-regulated and 6 miRNAs were up-regulated. Furthermore, 13 DE miRNAs putatively regulated 614 DE mRNAs. Among them, 20 important anther genes were predicted as target genes of MIR319A, MIR447A, MIR447B and MIR398B, respectively. Over-expression MIR319A and MIR447A could effectively inhibit the transcription of target genes and lead to male sterile. It suggested that DE miRNAs might mediate temperature signals and regulate anther and pollen development. Our work will provide a broader idea and valuable data information for further understanding the mechanism of thermo-sensitive male fertility in plants.

Utilizing Potato Virus X to Monitor RNA Movement.

Mobility assays coupled with RNA profiling have revealed the presence of hundreds of full-length non-cell-autonomous messenger RNAs that move through the whole plant via the phloem cell system. Monitoring the movement of these RNA signals can be difficult and time consuming. Here we describe a simple, virus-based system for surveying RNA movement by replacing specific sequences within the viral RNA genome of potato virus X (PVX) that are critical for movement with other sequences that facilitate movement. PVX is a RNA virus dependent on three small proteins that facilitate cell-to-cell transport and a coat protein (CP) required for long-distance spread of PVX. Deletion of the CP blocks movement, whereas replacing the CP with phloem-mobile RNA sequences reinstates mobility. Two experimental models validating this assay system are discussed. One involves the movement of the flowering locus T RNA that regulates floral induction and the second involves movement of StBEL5, a long-distance RNA signal that regulates tuber formation in potato.

De novo RNA sequencing analysis of Aeluropus littoralis halophyte plant under salinity stress.

The study of salt tolerance mechanisms in halophyte plants can provide valuable information for crop breeding and plant engineering programs. The aim of the present study was to investigate whole transcriptome analysis of Aeluropus littoralis in response to salinity stress (200 and 400 mM NaCl) by de novo RNA-sequencing. To assemble the transcriptome, Trinity v2.4.0 and Bridger tools, were comparatively used with two k-mer sizes (25 and 32 bp). The de novo assembled transcriptome by Bridger (k-mer 32) was chosen as final assembly for subsequent analysis. In general, 103290 transcripts were obtained. The differential expression analysis (log2FC > 1 and FDR < 0.01) showed that 1861 transcripts expressed differentially, including169 up and 316 down-regulated transcripts in 200 mM NaCl treatment and 1035 up and 430 down-regulated transcripts in 400 mM NaCl treatment compared to control. In addition, 89 transcripts were common in both treatments. The most important over-represented terms in the GO analysis of differentially expressed genes (FDR < 0.05) were chitin response, response to abscisic acid, and regulation of jasmonic acid mediated signaling pathway under 400 mM NaCl treatment and cell cycle, cell division, and mitotic cell cycle process under 200 mM treatment. In addition, the phosphatidylcholine biosynthetic process term was common in both salt treatments. Interestingly, under 400 mM salt treatment, the PRC1 complex that contributes to chromatin remodeling was also enriched along with vacuole as a general salinity stress responsive cell component. Among enriched pathways, the MAPK signaling pathway (ko04016) and phytohormone signal transduction (ko04075) were significantly enriched in 400 mM NaCl treatment, whereas DNA replication (ko03032) was the only pathway that significantly enriched in 200 mM NaCl treatment. Finally, our findings indicate the salt-concentration depended responses of A. littoralis, which well-known salinity stress-related pathways are induced in 400 mM NaCl, while less considered pathways, e.g. cell cycle and DNA replication, are highlighted under 200 mM NaCl treatment.

Long non-coding RNAs in the alkaline stress response in sugar beet (Beta vulgaris L.).

BACKGROUND: Long noncoding RNAs (lncRNAs) play crucial roles in regulating numerous biological processes in which complicated mechanisms are involved. Nonetheless, little is known about the number, features, sequences, and possible effects of lncRNAs on plant responses to alkaline stress. RESULTS: Leaf samples collected based on the control Beta vulgaris L., as well as those under short-term and long-term alkaline treatments, were subjected to high-throughput RNA sequencing, through which a total of 8535 lncRNAs with reliable expression were detected. Of these lncRNAs, 102 and 49 lncRNA expression profiles were altered after short- and long-term alkaline stress, respectively. Moreover, 7 lncRNAs were recognized as precursors to 17 previously identified miRNAs. Four lncRNAs responsive to alkaline stress were estimated as targets for 8 miRNAs. Moreover, computational analysis predicted 4318 potential target genes as lncRNAs responsive to alkaline stress. Analysis of functional annotations showed that the abovementioned possible target genes were involved in various bioprocesses, such as kinase activity, structural constituents of ribosomes, the ribonucleoprotein complex and protein metabolic processes. Association analysis provided convincing proof of the interplay of specific candidate target genes with lncRNAs. CONCLUSION: LncRNAs likely exert vital roles during the regulation of the alkaline stress response and adaptation in plants through interaction with protein-coding genes. The findings of this study contribute to comprehensively examining lncRNAs in Beta vulgaris L. and shed more light on the possible roles and modulating interplays of lncRNAs responsive to alkaline stress, thereby laying a certain basis for functional analyses of these types of Beta vulgaris L. lncRNAs in the future.

A novel insight into nitrogen and auxin signaling in lateral root formation in tea plant [Camellia sinensis (L.) O. Kuntze].

BACKGROUND: Tea plant (Camellia sinensis) is one of the most popular non-alcoholic beverages worldwide. In tea, lateral roots (LRs) are the main organ responsible for the absorption of moisture and mineral nutrients from the soil. Lateral roots formation and development are regulated by the nitrogen and auxin signaling pathways. In order to understand the role of auxin and nitrogen signaling in LRs formation and development, transcriptome analysis was employed to investigate the differentially expressed genes involved in lateral roots of tea plants treated with indole-3-butyric acid (IBA), N-1-naphthylphthalamic acid (NPA), low and high concentrations of nitrogen. RESULTS: A total of 296 common differentially expressed genes were identified and annotated to four signaling pathways, including nitrogen metabolism, plant hormone signal transduction, glutathione metabolism and transcription factors. RNA-sequencing results revealed that majority of differentially expressed genes play important roles in nitrogen metabolism and hormonal signal transduction. Low nitrogen condition induced the biosynthesis of auxin and accumulation of transcripts, thereby, regulating lateral roots formation. Furthermore, metabolism of cytokinin and ethylene biosynthesis were also involved in lateral roots development. Transcription factors like MYB genes also contributed to lateral roots formation of tea plants through secondary cell wall biosynthesis. Reversed phase ultra performance liquid chromatography (RP-UPLC) results showed that the auxin concentration increased with the decreased nitrogen level in lateral roots. Thus, tea plant lateral roots formation could be induced by low nitrogen concentration via auxin biosynthesis and accumulation. CONCLUSION: This study provided insights into the mechanisms associated with nitrogen and auxin signaling pathways in LRs formation and provides information on the efficient utilization of nitrogen in tea plant at the genetic level.

Integrated transcriptome and microRNA profiles analysis reveals molecular mechanisms underlying the consecutive monoculture problem of Polygonatum odoratum.

Polygonatum odoratum is a historically traditional Chinese medicine plant. However, the consecutive monoculture problem (CMP) widespread in other Chinese medicine limiting their cultivation on a large scale. In this study, the physiological data showed the adverse effect of CMP on the growth of P. odoratum under the consecutive cropping (CC) compared with the first cropping (FC). Then the high-throughput sequencing of miRNA and mRNA libraries of leaves and roots from FC and CC P. odoratum plants identified 671 differentially expressed genes (DEGs) and 184 differentially expressed miRNAs and revealed that the DEGs and target genes of the miRNAs were mainly involved in starch and sucrose metabolism, phenylpropanoid and brassinosteroid biosynthesis. The KEGG analysis revealed that the DEGs between CC and FC roots were enriched in the plant-pathogen interaction pathway. This study provided the expression regulation of genes related to CMP of P. odoratum but also suggested that CMP may result in the serious damage of pathogens to roots and cause the slow growth in the consecutive cropping plants.

MicroRNA160 regulates leaf curvature in potato (Solanum tuberosum L. cv. Désirée).

Leaf development is a complex process and factors such as size, shape, curvature, compounding, and texture determine the final leaf morphology. MicroRNA160 is one of the crucial players that has been shown to regulate lamina formation and compounding in tomato. In this study, we show that miR160 also regulates leaf curvature in potato. miR160 targets a group of Auxin Response Factors - StARF10, StARF16, and StARF17 - that are proposed to function majorly as repressors of auxin signaling. We observed that overexpression of miR160 (miR160-OE) results in decrease in the levels of these ARFs along with hypersensitivity to exogenous auxin treatment, whereas knockdown of miR160 (miR160-KD) causes increased ARF levels and auxin hyposensitivity. The leaves of miR160-OE plants have a high positive curvature, but of miR160-KD plants are flattened compared to wildtype. A prolonged activation of cell cycle - as indicated by increased levels of StCYCLIND3;2 - in the center region of miR160-OE leaves appears to have caused this positive curvature. However, a comparable StTCP4 activity at both center and margin regions of miR160-KD leaves could be the cause for its flattened leaf phenotype. In summary, we show that miR160 plays an important role in regulating leaf curvature in potato plants.

RNA-Seq based transcriptome analysis reveals the molecular mechanism of triterpenoid biosynthesis in Glycyrrhiza glabra.

Licorice is a frequently-used medicinal plant worldwide. Two triterpenoids, 18α-glycyrrhizic acid (18α-GC) and 18ß-glycyrrhizic acid (18ß-GC), are the key medicinal components accumulated in licorice. Biosynthesis of triterpenoids is a complex process that involves many secondary metabolic pathways. In this study, we tried to identify the key enzymes and pathways for the triterpenoid biosynthesis in licorice by analyzing the gene expression patterns in samples containing different GC levels. Glycyrrhizia glabra (one of the original species used as licorice in Chinese Pharmacopoeia) seeds were irradiated by X-ray and cultivated for one year, and samples with different GC contents were selected by HPLC analysis. RNA-Seq was performed to determine the gene expression in three X-ray irradiated G. glabra samples (H1, H2, and H3) with the highest GC content and one control G. glabra sample (L1) with the lowest GC content. 28.44 Gb raw data was generated and 47.7 million, 45.4 million, 43.3 million, and 45.9 million clean reads were obtained in samples H1, H2, H3, and L1, respectively. Approximately 48.53% of genes were annotated searching against GO and KEGG databases. A total of 1376 core differentially expressed genes (DEGs) were identified, which mainly enriched in phenylpropanoid metabolism, glycometabolism, plant circadian rhythm, and terpenoid biosynthetic pathway. 15 core DEGs selected from the 1376 DEGs were further verified by qRT-PCR, which confirmed that the RNA-Seq results were accurate and reliable. This study provides a basis for future functional genes mining and molecular regulatory mechanism elucidation of triterpenoid biosynthesis in licorice.