Integrated analysis of transcriptome and miRNAome reveals the heat stress response of Pinellia ternata seedlings.

Pinellia ternata (Thunb.) Briet., a valuable herb native to China, is susceptible to the "sprout tumble" phenomenon because of high temperatures, resulting in a significant yield reduction. However, the molecular regulatory mechanisms underlying the response of P. ternata to heat stress are not well understood. In this study, we integrated transcriptome and miRNAome sequencing to identify heat-response genes, microRNAs (miRNAs), and key miRNA-target pairs in P. ternata that differed between heat-stress and room-temperature conditions. Transcriptome analysis revealed extensive reprogramming of 4,960 genes across various categories, predominantly associated with cellular and metabolic processes, responses to stimuli, biological regulation, cell parts, organelles, membranes, and catalytic and binding activities. miRNAome sequencing identified 1,597 known/conserved miRNAs that were differentially expressed between the two test conditions. According to the analysis, genes and miRNAs associated with the regulation of transcription, DNA template, transcription factor activity, and sequence-specific DNA binding pathways may play a major role in the resistance to heat stress in P. ternata. Integrated analysis of the transcriptome and miRNAome expression data revealed 41 high-confidence miRNA-mRNA pairs, forming 25 modules. MYB-like proteins and calcium-responsive transcription coactivators may play an integral role in heat-stress resistance in P. ternata. Additionally, the candidate genes and miRNAs were subjected to quantitative real-time polymerase chain reaction to validate their expression patterns. These results offer a foundation for future studies exploring the mechanisms and critical genes involved in heat-stress resistance in P. ternata.

Comprehensive in silico characterization of NAC transcription factor family of Pinellia ternata and functional analysis of PtNAC66 under high-temperature tolerance in transgenic Arabidopsis thaliana.

Pinellia ternata, a valuable Chinese herb, suffers yield reduction due to "sprout tumble" under high temperatures. However, the mechanisms underlying its high-temperature stress remain poorly understood. NAM, ATAF1/2, and CUC2 (NAC) transcription factors regulate plant tissue growth and abiotic stress. Hence, there has been no comprehensive research conducted on NAC transcription factors in P. ternata. We identified 98 PtNAC genes unevenly distributed across 13 chromosomes, grouped into 15 families via phylogenetic analysis. Gene expression analysis revealed diverse expression patterns of PtNAC genes in different tissue types. Further studies revealed that PtNAC5/7/17/35/43/47/57/66/86 genes were highly expressed in various tissues of P. ternata and induced by heat stress, among which PtNAC66 was up-regulated at the highest folds induced by heat temperature. PtNAC66 is a nuclear protein that can selectively bind to the cis-responsive region NACRS but lacks the ability to activate transcription in yeast. For further research, PtNAC66 was cloned and transgenic Arabidopsis was obtained. PtNAC66 overexpression increased high-temperature tolerance compared to wild-type plants. Transcriptome profiling demonstrated that overexpression of PtNAC66 led to significant modification of genes responsible for regulating binding, catalytic activity, transcription regulator activity and transporter activity response genes. Additionally, PtNAC66 was found to bind the promoters of CYP707A3, MYB102 and NAC055, respectively, and inhibited their expression, affecting the high-temperature stress response in Arabidopsis. Our research established the foundation for functional studies of PtNAC genes in response to high-temperature forcing by characterizing the P. ternata NAC gene family and examining the biological role of PtNAC66 in plant high-temperature tolerance.

Assembly and comparative analysis of the complete mitochondrial genome of Pinellia ternata.

Pinellia ternata is an important natural medicinal herb in China. However, it is susceptible to withering when exposed to high temperatures during growth, which limits its tuber production. Mitochondria usually function in stress response. The P . ternata mitochondrial (mt) genome has yet to be explored. Therefore, we integrated PacBio and Illumina sequencing reads to assemble and annotate the mt genome of P . ternata . The circular mt genome of P . ternata is 876 608bp in length and contains 38 protein-coding genes (PCGs), 20 tRNA genes and three rRNA genes. Codon usage, sequence repeats, RNA editing and gene migration from chloroplast (cp) to mt were also examined. Phylogenetic analysis based on the mt genomes of P . ternata and 36 other taxa revealed the taxonomic and evolutionary status of P . ternata . Furthermore, we investigated the mt genome size and GC content by comparing P . ternata with the other 35 species. An evaluation of non-synonymous substitutions and synonymous substitutions indicated that most PCGs in the mt genome underwent negative selection. Our results provide comprehensive information on the P . ternata mt genome, which may facilitate future research on the high-temperature response of P . ternata and provide new molecular insights on the Araceae family.

A novel Pinellia ternata catalase gene PtCAT2 regulates drought tolerance in Arabidopsis by modulating ROS balance.

Drought is one of the major abiotic stresses limiting agricultural production, particularly for shallow-rooted plants like Pinellia ternata. It damages plants via oxidative burst, but this effect could be mitigated by catalase (CAT). However, no studies have been reported on CAT homologs in P. ternata, a drought-sensitive plant species. In the present study, a novel CAT gene, PtCAT2, was functionally characterized via overexpression in Arabidopsis and analysis of cis-elements in its promoter. The isolated CAT gene was 1479 bp and encoded a protein containing 242 amino acids. The protein contains the CAT activity motif and the heme-binding site of a typical CAT, and the subcellular analysis indicated that the protein localizes at the cytoplasm and membrane. Moreover, the quantitative real-time reverse transcription PCR indicated that PtCAT2 is expressed ubiquitously in P. ternata and is strongly induced by drought stress and abscisic acid (ABA) signals. PtCAT2 overexpression enhanced the drought tolerance of Arabidopsis, as shown by the 30% increase in plant survival and a five-fold- increase in CAT activity. Moreover, PtCAT2-transgenic plants increased superoxide dismutase and peroxidase activities and reduced malondialdehyde, membrane leakage, and hydrogen peroxide (H2O2) (P<0.05). Furthermore, PtCAT2-transgenic plants showed higher tolerance to oxidative stress caused by exogenous H2O2 and retained higher chlorophyll and water contents than the WT. The mitochondria function was better maintained as presented by the higher oxygen consumption rate in transgenics under drought stress (P<0.05). The endogenous CATs and drought response-related genes were also upregulated in transgenic lines under drought stress, indicating that PtCAT2 confers drought stress tolerance by enhancing the H2O2 scavenging ability of plants to maintain their membrane integrity. These results improve our understanding of the drought response mechanisms and provide a potential breeding strategy for P. ternata genetic improvement.

Transcriptome Analysis Reveals Long Non-Coding RNAs Involved in Shade-Induced Growth Promotion in Pinellia ternata.

BACKGROUND: High temperature and drought environments are important limiting factors for Pinellia ternata growth, whereas shading can promote growth by relieving these stresses. However, the mechanism of growth promotion by shading in P. ternata is unknown. Long non-coding RNAs (lncRNAs) play important roles in the plant's growth and environmental response, but few analyses of lncRNAs in P. ternata have been reported. METHODS: We performed lncRNAs analysis of P. ternata in response to shading using RNA-seq data from our previous studies. A total of 13,927 lncRNAs were identified, and 145 differentially expressed lncRNAs (DELs) were obtained from the comparisons of 5 days shade (D5S) vs. 5 days of natural light (D5CK), 20 days of shade (D20S) vs. 20 days of natural light (D20CK), D20S vs. D5S, and D20CK vs. D5CK. Of these, 119 DELs (82.07%) were generated from the D20S vs. D20CK comparison. RESULTS: Gene ontology (GO) analysis indicated that the reactive oxygen (ROS) metabolism and programmed cell death (PCD) processes might regulate shade-induced growth promotion. The "signal transduction" and "environmental adaptation" in the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were used for lncRNA-mRNA regulatory network construction and showed that the lncRNAs might mediate P. ternata growth by regulating ROS accumulation and light signals. CONCLUSIONS: This study explores lncRNAs' functions and regulatory mechanisms related to P. ternata growth and lays a foundation for further research on P. ternata.

Extraocular muscle volume index at the orbital apex with optic neuritis: a combined parameter for diagnosis of dysthyroid optic neuropathy.

OBJECTIVES: To evaluate the diagnostic performance of the extraocular muscle volume index at the orbital apex (AMI) and the signal intensity ratio (SIR) of the optic nerve in dysthyroid optic neuropathy (DON). METHODS: Clinical data and magnetic resonance imaging were collected retrospectively from 63 Graves' ophthalmopathy patients, including 24 patients with DON and 39 without DON. The volume of these structures was obtained by reconstructing their orbital fat and extraocular muscles. The SIR of the optic nerve and axial length of eyeball were also measured. The posterior 3/5 of the retrobulbar space volume was used as the orbital apex to compare parameters in patients with or without DON. Area under the receiver operating characteristic curve (AUC) analysis was used to select the morphological and inflammatory parameters with the highest diagnostic value. A logistic regression was performed to identify the risk factors of DON. RESULTS: One hundred twenty-six orbits (35 with DON and 91 without DON) were analyzed. Most of the parameters in DON patients were significantly higher than in non-DON patients. However, the SIR 3 mm behind the eyeball of the optic nerve and AMI had the highest diagnostic value in these parameters and are independent risk factors of DON by stepwise multivariate logistic regression analysis. Combining AMI and SIR had a higher diagnostic value than a single index. CONCLUSIONS: Combining AMI with SIR 3 mm behind the eyeball's orbital nerve can be a potential parameter for diagnosing DON. CLINICAL RELEVANCE STATEMENT: The present study provided a quantitative index based on morphological and signal changes to assess the DON, allowing clinicians and radiologists to monitor DON patients timely. KEY POINTS: The extraocular muscle volume index at the orbital apex (AMI) has excellent diagnostic performance for dysthyroid optic neuropathy. A signal intensity ratio (SIR) of 3 mm behind the eyeball has a higher AUC compared to other slices. Combining AMI and SIR has a higher diagnostic value than a single index.

Choriocapillaris and progressive ganglion cell-inner plexiform layer loss in non-glaucomatous eyes.

AIMS: To explore the relationship between choriocapillaris (CC) flow deficit percentage (FD%) and ganglion cell-inner plexiform layer (GCIPL) thickness in a population-based sample of non-glaucomatous eyes. METHODS: This is a longitudinal cohort study and prospective cross-sectional study. Non-glaucoma Chinese subjects aged 18 years or older were enrolled. All participants underwent a detailed ophthalmic examination, including swept-source optical coherence tomography (SS-OCT) and SS-OCT angiography. Average, inner average, outer average and nine Early Treatment Diabetic Retinopathy Study sub-regions of GCIPL thickness and CC FD% were measured. The correlation between CC FD% and GCIPL was assessed using a linear regression model, and the relationship between the rate of change of GCIPL thickness and CC FD% was further validated in a 2year longitudinal study. RESULTS: In the cross-sectional study including 3514 participants (3514 non-glaucoma eyes), a higher CC FD% was significantly associated with a thinner GCIPL (ß=-0.32; 95% CI -0.43 to -0.21; p<0.001). Further, in a longitudinal study (453 eyes of 453 participants), a faster increase in CC FD% was found to be significantly associated with a faster decrease in GCIPL thickness (ß=-0.10; 95% CI -0.17 to -0.03; p=0.004) after adjusting for age, sex, axial length and image quality score. CONCLUSIONS: This is the first time to show that CC FD% and GCIPL thickness were correlated in both cross-sectional and longitudinal studies of non-glaucomatous individuals, which may potentially provide further insights on the role of CC perfusion in glaucoma development and progression.

Ultra-low concentration of chlorine dioxide regulates stress-caused premature leaf senescence in tobacco by modulating auxin, ethylene, and chlorophyll biosynthesis.

Exploring novel growth regulators for premature senescence regulation is important for tobacco production. In the present study, chlorine dioxide (ClO2) was explored as a novel plant growth regulator for tobacco growth, particularly its effect on leaf senescence and root development. The results showed that 0.15 µM ClO2 maintained the lushness of detached leaves and whole plants. Also, the leaves of ClO2-treated plants exhibited a chlorophyll content of 58% higher than in CK (control) plants (P < 0.05). Besides, ClO2 treatment increased the biomass of roots and aboveground parts by 54 and 16%, respectively. The ClO2-treated plants also showed enhanced activities of antioxidant enzymes and significantly reduced malondialdehyde contents (P < 0.05). Moreover, ClO2 treatment remarkably alleviated drought-caused premature senescence in the tobacco plants and partly rescued the exogenous ethylene-caused plant dwarfism. The indole-3-acetic acid content in ClO2-treated plants was higher than in non-treated plants (P < 0.05), but ethylene content was significantly lower (P < 0.05). Gene expression analysis showed that ClO2 treatment remarkably suppressed ethylene synthase genes. However, the auxin biosynthesis and transport genes were up-regulated, with NtIAA17 increasing by five folds (P < 0.05). Further, ClO2 remarkably up-regulated the expression of chlorophyll biosynthesis genes, with a >20-fold increase in NtHEMA1 and NtCHLH expressions. These results designate ClO2 as a potential regulator for improving tobacco productivity by retaining higher chlorophyll content and promoting root growth.

Functional characterization of the Pinellia ternata cytoplasmic class II small heat shock protein gene PtsHSP17.2 via promoter analysis and overexpression in tobacco.

High temperature is one of the main abiotic factors limiting agricultural production, particularly for heat-sensitive plant species. Small heat-shock proteins contribute substantially to alleviating damage to plants caused by heat stress. In the present study, the heat shock protein gene PtsHSP17.2 from Pinellia ternata was functionally characterized through promoter analysis and its overexpression in tobacco. Respectively, relative expression using real-time RT-PCR and ex situ promoter activity assay indicated that PtsHSP17.2 is strongly inducible under heat stress, and in silico promoter analysis discovered multiple stress-related cis elements including heat shock element. When overexpressing PtsHSP17.2 in tobacco, the thermotolerance of transgenic plants was markedly enhanced. Furthermore, the transgenic tobacco plants exhibited less variation in chlorophyll content, relative electrolyte leakage, and malondialdehyde content under heat stress compared with wild-type (WT) plants. The activities of antioxidant enzymes and content of proline were significantly enhanced under heat stress in transgenic plants relative to WT plants. Transgenic plants also had enhanced water retention and increased antioxidative capacity. Further, the expression levels of genes encoding antioxidant enzymes were more highly induced by heat stress in transgenic lines than WT. These results enrich the current understanding of thermal adaptation of heat-sensitive plant species and encourage further genetic improvement.

Analysis of DNA methylation in potato tuber in response to light exposure during storage.

Exposure to light induces tuber greening and the accumulation of the toxic alkaloid Solanine in potato (Solanum tuberosum L) during storage greatly reduce tuber value. While the mechanism of this greening process remains unclear, it is well understood that DNA methylation plays an important role in regulating gene expression in response to environmental conditions. In this study, methylation-sensitive amplified polymorphism was used to assess the effect of light exposure on DNA methylation during storage of potato tubers. Light-induced genome-wide DNA demethylation and the rate of DNA methylation decreased with long storage times. Following, the sequencing of 14 differentially amplified fragments and analysis using the Basic Local Alignment Search Tool, eight genomic sequences and six annotated fragment sequences were identified. The latter included ADP glucose pyrophosphorylase 1/2, chlorophyllide a oxygenase 1 (CAO1), receptor-like protein kinase HAIKU2, and repressor of GA4, all of which are involved in starch biosynthesis, chlorophyll synthesis, endosperm development, and gibberellic acid signaling, respectively. Demethylation was observed in the CpG island (-273 to -166 bp) of the CAO1 promoter in response to light, which further confirmed that the variations in genome methylation are dependent upon the light exposure and suggests a direct role for DNA methylation. Our results provide an epigenetic perspective for further exploring the mechanism of light-induced tuber greening.

UHPLC-MS-based metabolomic approach for the quality evaluation of Pinellia ternata tubers grown in shaded environments.

Pinellia ternata is a native herb in China, and its tuber is widely-used in traditional Chinese medicines. It has been identified that the shading treatment promotes tuber production during cultivation. However, the tuber quality in shaded environments is unknown, which limits the scientific cultivation of P. ternata. In this study, a metabolomics approach based on UHPLC-MS was applied to assess the metabolic components of P. ternata in response to shading. Diverse metabolites were profiled using the metabolomics approach. Then, datasets of P. ternata cultivated in natural light (control) and shaded environments were subjected to multivariate analyses. Two P. ternata tuber products were well separated by the PCA. In total, four P. ternata alkaloids with contents that were not altered by the shaded environment were detected. Metabolomic analyses further identified several organic acids [mevalonic acid, 12,13-dihydroxy-9Z-octadecenoic acid (12, 13-DiHOME), urocanic acid, and γ-aminobutyric acid] that were largely enriched in the shaded environment, which likely contributed to tuber quality and growth. This study determined that shading probably improves the quality of P. ternata tubers and laid a foundation for exploring the regulatory mechanism of the shade response in P. ternata.

Genotypic variation of flavonols and antioxidant capacity in broccoli.

Flavonols are gaining increasing interests due to their diverse health benefits for humans. Broccoli is a main flavonol source in our diet, but the genetic variation of flavonols and their correlation with antioxidant capacity remain to be understood. Here, we examined variations of the two major flavonols kaempferol and quercetin in florets and leaves of 15 diverse broccoli accessions by ultra-performance liquid chromatography. Broccoli accumulated more kaempferol than quercetin in most of the accessions tested, with the ratios varying from 4.4 to 27.9 in leaves and 0.4 to 4.4 in florets. Total flavonoids showed 2.5-fold and 3.3-fold differences in leaves and florets of these accessions, respectively. Principle component analysis revealed that flavonols, along with the key biosynthetic pathway genes, correlated with antioxidant capacity related indicators. This study provides important information for broccoli flavonol genotypic variations and correlation with antioxidant capacity, and will facilitate the development of flavonol enriched cultivars in broccoli.

Biochemical basis of differential selenium tolerance in arugula (Eruca sativa Mill.) and lettuce (Lactuca sativa L.).

Selenium (Se) biofortification in crops provides a valuable strategy to enhance human Se intake. However, crops vary greatly with their capacity in tolerating and metabolizing/accumulating Se, and the basis underlying such variations remains to be fully understood. Here, we compared the effects of Se and its analog S treatments on plant growth and biochemical responses between a Se accumulator (arugula) and a non-accumulator (lettuce). Arugula exhibited an increased biomass production in comparison with untreated controls at a higher selenate concentration than lettuce (20 µM vs. 10 µM Na2SeO4), showing better tolerance to Se. Arugula accumulated 3-folds more Se and S than lettuce plants under the same treatments. However, the Se/S assimilation as assessed by ATP sulfurylase and O-acetylserine (thiol)lyase activities was comparable between arugula and lettuce plants. Approximately 4-fold higher levels of Se in proteins under the same doses of Se treatments were observed in arugula than in lettuce, indicating that Se accumulators have better tolerance to selenoamino acids in proteins. Noticeably, arugula showed 6-fold higher ascorbate peroxidase activity and produced over 5-fold more glutathione and non-protein thiols than lettuce plants, which suggest critical roles of antioxidants in Se tolerance. Taken together, our results show that the elevated Se tolerance of arugula compared to lettuce is most likely due to an efficient antioxidant defense system. This study provides further insights into our understanding of the difference in tolerating and metabolizing/accumulating Se between Se accumulators and non-accumulators.

Full-length transcriptome analysis of shade-induced promotion of tuber production in Pinellia ternata.

BACKGROUND: Pinellia ternata is native to China and has been used as a traditional herb due to its antiemetic, antitussive, analgesic, and anxiolytic effects. When exposed to strong light intensity and high temperature during the reproductive growth process, P. ternata withers in a phenomenon known as "sprout tumble", which largely limits tuber production. Shade was previously found to delay sprout tumble formation (STF); however, no information exists regarding this process at the molecular level. Hence, we determined the genes involved in tuber development and STF in P. ternata. RESULTS: Compared to that with natural sun-light (control), shade significantly induced chlorophyll accumulation, increased chlorophyll fluorescence parameters including initial fluorescence, maximal fluorescence, and qP, and dramatically repressed chlorophyll a:b and NPQ. Catalase (CAT) activity was largely induced by shade, and tuber products were largely increased in this environment. Transcriptome profiles of P. ternata grown in natural sun-light and shaded environments were analyzed by a combination of next generation sequencing (NGS) and third generation single-molecule real-time (SMRT) sequencing. Corrections of SMRT long reads based on NGS short reads yielded 136,163 non-redundant transcripts, with an average N50 length of 2578 bp. In total, 6738 deferentially-expressed genes (DEGs) were obtained from the comparisons, specifically D5S vs D5CK, D20S vs D20CK, D20S vs D5S, and D20CK vs D5CK, of which, 6384 DEGs (94.8%) were generated from the D20S vs D20CK comparison. Gene annotation and functional analyses revealed that these genes were related to auxin signal transduction, polysaccharide and sugar metabolism, phenylpropanoid biosynthesis, and photosynthesis. Moreover, the expression of genes enriched in photosynthesis appeared to be significantly altered by shade. The expression patterns of 16 candidate genes were consistent with changes in their transcript abundance as identified by RNA-Seq, and these might contribute to STF and tuber production. CONCLUSION: The full-length transcripts identified in this study have provided a more accurate depiction of P. ternata gene transcription. Further, we identified potential genes involved in STF and tuber growth. Such data could serve as a genetic resource and a foundation for further research on this important traditional herb.

Use of chlorine dioxide to sterilize medium for tissue culture of potato.

In vitro cultured seedlings or microtubers are the major starting materials for the production of potato. Currently, seedlings are cultured in media sterilized by autoclaving, which, however, consumes more electricity and takes longer for sterilization, and also requires high temperature-tolerant vessel materials. In order to identify alternative methods of sterilizing culture conditions, the disinfection effects of chlorine dioxide (CD) at 88.0, 29.3, 17.6, 12.6 and 8.8 µM were evaluated in potato medium and vessels. The ≥12.6 µM gaseous CD effectively disinfected vessel through a 30-min fumigation process, and its aqueous solution disinfected potato medium efficiently as well. In presence of 12.6 µM CD in the medium, the potato seedlings had similar morphological features as those grown on autoclaved medium, with some exceptions. The use of 12.6-29.3 µM aqueous CD to sterilize the medium increased antioxidant enzyme activities in potato seedlings, while the use of higher concentration decreased antioxidant enzyme activity levels. SSR analysis did not reveal significant molecular differences in potato seedlings cultured between autoclaved and CD-sterilized medium. In addition to this, CD-sterilized medium induced potato microtuber formation at a similar rate as autoclaved medium. In summary, using CD to sterilize potato medium and vessels did not compromise the growth of seedlings and microtuber induction. This study provides an economical and simplified sterilization method for media used to culture potato plantlets, and this can improve energy use of the large-scale tissue culture industry.

Effect of soil types and ammonia concentrations on the contribution of ammonia-oxidizing bacteria to CH4 oxidation.

Irrigation of stabilized landfill leachate to landfill cover soil is a cost-effective operation for leachate treatment. The contribution of ammonia-oxidizing bacteria (AOB) in the cover soil to CH4 oxidation, however, is unclear, because AOB and methane-oxidizing bacteria (MOB) can co-oxidize CH4 and NH4+-N. Thus, the contribution of AOB and the inhibitory effect of NH4+-N to CH4 oxidation were determined by using an acetylene pretreatment discrimination method. The results showed that the contributions of AOB to CH4 oxidation varied with the soil type and the concentration of NH4+-N addition. The relative contribution of AOB to CH4 oxidation for compost without NH4+-N addition was the highest (65.0%), and was 2.5 and 3.4 times higher than the corresponding values for aged refuse and landfill cover soil, respectively. The inhibitory effect of NH4+-N was enhanced by increasing the concentration of NH4+-N addition for all the soil samples. At equal NH4+-N addition concentrations, the inhibitory effect was always the lowest for the compost sample. The abundances of particulate methane monooxygenase (pmoA) and ammonia monooxygenase (amoA) genes were key factors influencing the CH4 oxidation rate and contribution of AOB to CH4 oxidation. The higher abundance of pmoA and lower abundance of amoA in landfill cover soil could explain the higher CH4 oxidation rate and lower contribution of AOB to CH4 oxidation in this soil type. Meanwhile, the higher contribution of AOB to CH4 oxidation for compost could be attributed to the higher abundance of the amoA gene and lower abundance of pmoA.

Comparative transcriptome analysis of roots, stems and leaves of Isodon amethystoides reveals candidate genes involved in Wangzaozins biosynthesis.

BACKGROUND: Isodon amethystoides (Ben-th) Cy Wu et Hsuan is an important traditional medicinal plant endowed with pharmacological properties effective in the treatment of various diseases, including pulmonary tuberculosis. The tetracyclic diterpenoids, Wangzaozins (Wangzaozin A, glaucocalyxin A, glaucocalyxin B), are the major bioactive compounds of I. amethystoides. However, the molecular information about the biosynthesis of these compounds still remains unclear. RESULTS: An examination of the accumulated levels of Wangzaozins in I. amethystoides revealed considerable variations in the root, stem, and leaf tissues of this plant, indicating possible differences in metabolite biosynthesis and accumulation among various tissues. To better elucidate the tetracyclic diterpenoid biosynthesis pathway, we generated transcriptome sequences from the root, stem, and leaf tissues, and performed de novo sequence assembly, yielding 230,974 transcripts and 114,488 unigenes, with average N50 lengths of 1914 and 1241 bp, respectively. Putative functions could be assigned to 73,693 transcripts (31.9%) based on BLAST searches against annotation databases, including GO, KEGG, Swiss-Prot, NR, and Pfam. Moreover, the candidate genes involving in the diterpenoid biosynthesis, such as CPS, KSL, were also analyzed. The expression profiles of eight transcripts, involving the tetracyclic diterpenoid biosynthesis, were validated in different I. amethystoides tissues by qRT-PCR, unraveling the gene expression profile of the pathway. The differential expressions of ISPD, ISPF and ISPH (MEP pathway), and IaCPS and IaKSL (diterpenoid pathway) candidate genes in leaves and roots, may contribute to the high accumulation of Wangzaozins in I. amethystoides leaves. CONCLUSION: The genomic dataset and analyses reported here lay the foundations for further research on this important medicinal plant.

[Identification of bear bile powder and its adulterants by using DNA barcoding technology].

To identify the precious bile powder and its adulterants by DNA barcoding, and establish its standard experimental process to ensure the safe and effective utilization. Total twelve sequences from samples of bear bile powder which come from Ursus thibetanus for DNA extraction, PCR(polymerase chain reaction) and sequence, then using CodonCode Aligner V 7.0.1 shear primer region to obtain COI sequence. The COI sequences of U. arctos and their adulterants were obtained from GenBank. MEGA7.0 software was applied for analyzing mutation, calculating intraspecific and interspecific K2P(Kimura 2-Parameter) genetic distance and constructing the Neighbor-joining tree(NJ). The results showed that the maximum K2P genetic distance of bear bile powder of U. thibetanus and U. arctos are far less than minimum K2P genetic distance within its adulterants species, and the results of NJ tree demonstrated that each species could be distinguished from the counterfeits obviously. DNA barcoding is a safe, convenient and reliable technique for species identification, and it is important to establish the standard sequence of COI sequences for animal medicines.

Identification of a regulatory element responsible for salt induction of rice OsRAV2 through ex situ and in situ promoter analysis.

Salt is a major environmental stress factor that can affect rice growth and yields. Recent studies suggested that members of the AP2/ERF domain-containing RAV (related to ABI3/VP1) TF family are involved in abiotic stress adaptation. However, the transcriptional response of rice RAV genes (OsRAVs) to salt has not yet been fully characterized. In this study, the expression patterns of all five OsRAVs were examined under salt stress. Only one gene, OsRAV2, was stably induced by high-salinity treatment. Further expression profile analyses indicated that OsRAV2 is transcriptionally regulated by salt, but not KCl, osmotic stress, cold or ABA (abscisic acid) treatment. To elucidate the regulatory mechanism of the stress response at the transcriptional level, we isolated and characterized the promoter region of OsRAV2 (P OsRAV2 ). Transgenic analysis indicated that P OsRAV2 is induced by salt stress but not osmotic stress or ABA treatment. Serial 5' deletions and site-specific mutations in P OsRAV2 revealed that a GT-1 element located at position -664 relative to the putative translation start site is essential for the salt induction of P OsRAV2 . The regulatory function of the GT-1 element in the salt induction of OsRAV2 was verified in situ in plants with targeted mutations generated using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system. Taken together, our results indicate that the GT-1 element directly controls the salt response of OsRAV2. This study provides a better understanding of the putative functions of OsRAVs and the molecular regulatory mechanisms of plant genes under salt stress.

Identification and utilization of cleistogamy gene cl7(t) in rice (Oryza sativa L.).

Gene transformation is an important method for improvement of plants into elite varieties. However, the possibility of gene flow between genetically modified (GM) crops and similar species is a serious public issue that may potentially endanger ecological stability. Cleistogamy is expected to be an ideal genetic tool for preventing transgene propagation from GM crops. A rice mutant, cl7(t), was created by ethyl methanesulfonate mutagenesis. The mutant exhibited cleistogamy, and had closed spikelets, reduced plant height, and altered morphology of the leaves, panicle, and seeds. Anatomical investigations revealed that the cl7(t) mutant contained more vascular bundles and thicker stems than the wild type, which increased the mechanical strength of its internodes, and anti-lodging ability. Further studies demonstrated that the force required to open the lemma and palea was higher in the cl7(t) mutant, and there was weak swelling ability in the lodicules, which leads to cleistogamy. Allelic analyses and complementation tests indicated that cl7(t) was a novel allele of dep2, a mutant that was previously reported to have similar panicle morphology. Sequence analysis showed that cl7(t) had a single nucleotide substitution (C to A) in the third exon that leads to a Ser substitution with a stop codon, giving a truncated DEP2 protein. Quantitative RT-PCR and in situ hybridization tests demonstrated that there was lower CL7(t) expression level in the spikelets and weaker CL7(t) signals in the lodicules of the cl7(t) mutant compared with wild type, which implies that CL7(t) might participate in the development of lodicules. To improve the agronomic traits of cl7(t) to fit the needs of field production, the cl7(t) mutant was crossed with an intermediate-type rice variety named Guanghui102, which bears some important agronomic traits, including increased grain numbers and high rate of seed setting. Through multi-generational pedigree selection, cleistogamy lines with improved economic traits were obtained, which can be used for the selection of ecologically safe GM rice varieties.