Comprehensive Analysis of Endogenous Volatile Compounds, Transcriptome, and Enzyme Activity Reveals PmCAD1 Involved in Cinnamyl Alcohol Synthesis in Prunus mume.

Floral scent is an important economic and ornamental trait of Prunus mume. The floral volatiles from most cultivars of P. mume in composition exist significant differences. Cinnamyl alcohol was one of the main floral volatile compounds with distinct abundances in different cultivars, namely, 'Zaohua Lve,' 'Zao Yudie,' 'Fenpi Gongfen,' 'Jiangsha Gongfen,' and 'Fenhong Zhusha.' Based on the determination of endogenous volatiles of full-blooming flowers, vital enzyme activity and transcriptomes were comprehensively analyzed to screen the key potential genes involved in cinnamyl alcohol synthesis. Transcriptome combining with enzyme activity level analysis suggested that the expression levels of three PmCADs were highly correlated with the cinnamyl alcohol dehydrogenase (CAD) enzyme activities in six cultivars. Furthermore, phylogenetic tree and transcriptome analysis suggested that PmCAD1 and PmCAD2 might contribute to the cinnamyl alcohol synthesis. Relative expression analyses and enzyme activity assays showed that PmCAD1 played an important role in cinnamyl alcohol biosynthesis in vitro. Overall, this research lays a theoretical foundation for clarifying comprehensively the molecular biosynthesis mechanism of floral volatiles in P. mume.

Characteristics and Expression Analyses of Trehalose-6-Phosphate Synthase Family in Prunus mume Reveal Genes Involved in Trehalose Biosynthesis and Drought Response.

Trehalose and its key synthase (trehalose-6-phosphate synthase, TPS) can improve the drought tolerance of plants. However, little is known about the roles of trehalose and the TPS family in Prunus mume response to drought. In our study, we discovered that the trehalose content in leaf, root, and stem tissues significantly increased in P. mume in response to drought. Therefore, the characteristics and functions of the TPS family are worth investigating in P. mume. We identified nine TPS family members in P. mume, which were divided into two sub-families and characterized by gene structure, promoter elements, protein conserved domains, and protein motifs. We found that the Hydrolase_3 domain and several motifs were highly conserved in Group II instead of Group I. The distinctions between the two groups may result from selective constraints, which we estimated by the dN/dS (ω) ratio. The ω values of all the PmTPS family gene pairs were evaluated as less than 1, indicating that purity selection facilitated their divergence. A phylogenetic tree was constructed using 92 TPSs from 10 Rosaceae species, which were further divided into five clusters. Based on evolutionary analyses, the five clusters of TPS family proteins mainly underwent varied purity selection. The expression patterns of PmTPSs under drought suggested that the TPS family played an important role in the drought tolerance of P. mume. Combining the expression patterns of PmTPSs and the trehalose content changes in leaf, stem, and root tissues under normal conditions and drought stress, we found that the PmTPS2 and PmTPS6 mainly function in the trehalose biosynthesis in P. mume. Our findings not only provide valuable information about the functions of trehalose and TPSs in the drought response of P. mume, but they also contribute to the future drought breeding of P. mume.

Overexpression of LiTPS2 from a cultivar of lily (Lilium 'Siberia') enhances the monoterpenoids content in tobacco flowers.

Lily, a famous cut flower with highly fragrance, has high ornamental and economic values. Monoterpenes are the main components contributing to its fragrance, and terpene synthase (TPS) genes play critical roles in the biosynthesis of monoterpenoids. To understand the function of TPS and to explore the molecular mechanism of floral scent in cultivar Lilium 'Siberia', transcriptomes of petal at different flowering stages and leaf were obtained by RNA sequencing and three unigenes related to TPS genes were selected for further validation. Quantitative real-time PCR showed that the expression level of LiTPS2 was greater than that of the other two TPS genes. Phylogenetic analysis indicated that LiTPS2 belonged to the TPSb subfamily, which was responsible for monoterpenes synthesis. Subcellular localization demonstrated that LiTPS2 was located in the chloroplasts. Furthermore, functional characterization showed that LiTPS2 utilized both geranyl pyrophosphate (GPP) and farnesyl pyrophosphate (FPP) to produce monoterpenoids such as linalool and sesquiterpenes like trans-nerolidol, respectively. Ectopic expression in transgenic tobacco plants suggested that the amount of linalool from the flowers of transgenic plants was 2-3 fold higher than that of wild-type plants. And the emissions of myrcene and (E)-ß-ocimene were also accumulated from the flowers of LiTPS2 transgenic lines. Surprisingly, these three compounds were the main fragrance components of oriental lily hybrids. Our results indicated that LiTPS2 contributed to the production of monoterpenes and could effectively regulate the aroma of Lilium cultivars, laying the foundation for biotechnological modification of floral scent profiles.

Root Physiological Traits and Transcriptome Analyses Reveal that Root Zone Water Retention Confers Drought Tolerance to Opisthopappus taihangensis.

Opisthopappus taihangensis (Ling) Shih, as a relative of chrysanthemum, mainly survives on the cracks of steep slopes and cliffs. Due to the harsh environment in which O. taihangensis lives, it has evolved strong adaptive traits to drought stress. The root system first perceives soil water deficiency, triggering a multi-pronged response mechanism to maintain water potential; however, the drought tolerance mechanism of O. taihangensis roots remains unclear. Therefore, roots were selected as materials to explore the physiological and molecular responsive mechanisms. We found that the roots had a stronger water retention capacity than the leaves. This result was attributed to ABA accumulation, which promoted an increased accumulation of proline and trehalose to maintain cell osmotic pressure, activated SOD and POD to scavenge ROS to protect root cell membrane structure and induced suberin depositions to minimize water backflow to dry soil. Transcriptome sequencing analyses further confirmed that O. taihangensis strongly activated genes involved in the ABA signalling pathway, osmolyte metabolism, antioxidant enzyme activity and biosynthesis of suberin monomer. Overall, these results not only will provide new insights into the drought response mechanisms of O. taihangensis but also will be helpful for future drought breeding programmes of chrysanthemum.

Identification of the epitope in human poliovirus type 1 Sabin strain recognized by the monoclonal antibody 1G10 using mimotope strategy.

Following the recommended use of the inactivated poliovirus vaccine from Sabin strains (sIPV) by the WHO, a D antigen-specific neutralizing monoclonal antibody (mAb) 1G10 that recognized the human poliovirus type 1 Sabin strain (PV-I Sabin) was produced for D-antigen potency evaluation on sIPV. Study of the mAb 1G10 showed that it recognized a discontinuous conformational epitope of PV-I Sabin antigen. To identify this epitope quickly, easily and cost-effectively, clues to the epitope's identity were first obtained by employing a novel mimotope strategy based on a phage display library and "in silico" prediction. Then, the conformation of the epitope region, including the amino acid sequence, neutralizing sites, and location of this epitope, was identified using several classic epitope-mapping methods such as synthesized peptides analysis, neutralization assay and site-directed mutagenesis. The mimotope strategy may offer some guidance for achieving epitope identification in a more feasible and effective way. This mAb could be used in an in-house or national and international standard IPV D-antigen potency ELISA kit in the future.

A Comparative Analysis of Floral Scent Compounds in Intraspecific Cultivars of Prunus mume with Different Corolla Colours.

Prunus mume is the only fragrant flowering species of Prunus. According to the previous studies, benzyl acetate and eugenol dominate its floral scent. However, the diversity of its floral scents remains to be elucidated. In this work, the floral volatiles emitted from eight intraspecific cultivars of P. mume with white, pink and red flowers, were collected and analyzed using headspace solid-phase microextraction combined with gas chromatograms-mass spectrometry (HS-SPME-GC-MS). In total, 31 volatile compounds were identified, in which phenylpropanoids/benzenoids accounted for over 95% of the total emission amounts. Surprisingly, except for benzyl acetate and eugenol, several novel components, such as benzyl alcohol, cinnamyl acohol, cinnamy acetate, and benzyl benzoate were found in some cultivars. The composition of floral volatiles in cultivars with white flowers was similar, in which benzyl acetate was dominant, while within pink flowers, there were differences of floral volatile compositions. Principal component analysis (PCA) showed that the emissions of benzyl alcohol, cinnamyl alcohol, benzyl acetate, eugenol, cinnamyl acetate, and benzyl benzoate could make these intraspecific cultivars distinguishable from each other. Further, hierarchical cluster analysis indicated that cultivars with similar a category and amount of floral compounds were grouped together. Our findings lay a theoretical basis for fragrant plant breeding in P. mume.

Safety of antivascular endothelial growth factor administration in the ocular anterior segment in pterygium and neovascular glaucoma treatment: Systematic review and meta-analysis.

BACKGROUND: Anti-VEGF agents has been widely used in ocular diseases, but its safety for treating anterior segment disorders, the conclusions are controversial. METHODS: Several major databases, including CENTRAL, MEDLINE, and EMBASE, were searched. Safety data from 18 randomized controlled trials (RCTs) were used to compare anti-VEGF treatment in the ocular anterior segment in pterygium and neovascular glaucoma treatment with placebo/sham treatment for eye diseases. A meta-analysis for adverse events was performed. RESULTS: Eighteen RCT studies with 955 eyes were included in the meta-analysis. Significant difference in conjunctival disorders (OR: 1.62; 95% CI, 1.01-2.59; P = .05) was noted among the included studies, but not in ocular intolerance (odds ratio [OR]: 0.75; 95% CI, 0.34-1.62; P = .46), corneal disorders (OR: 0.71; 95% CI, 0.37-1.37; P = .31), or the subgroup analysis of conjunctival disorders. CONCLUSIONS: The administration of anti-VEGF agents in the ocular anterior segment for patients with pterygium and glaucoma was tolerable in tolerance and cornea, but was the risk factor of conjunctival disorders. The healing of corneal epithelium may be delayed in patients with primary corneal epithelial defects after anti-VEGF application. However, due to the limited evidence, further research should be performed on the safety of anti-VEGF administration in patients with different corneal disorders.

Overexpression of LiDXS and LiDXR From Lily (Lilium 'Siberia') Enhances the Terpenoid Content in Tobacco Flowers.

Lilium, the famous and significant cut flower, emits a variety of volatile organic compounds, which mainly contain monoterpenes, such as myrcene, (E)-ß-ocimene, and linalool. To understand the molecular mechanism of monoterpene synthesis in Lilium, we cloned two potential genes in the methylerythritol 4-phosphate pathway, namely LiDXS and LiDXR, from the strong-flavored oriental Lilium 'Siberia' using a homology-based PCR strategy. The expression levels of LiDXS and LiDXR were consistent with the emission and accumulation of monoterpenes in different floral organs and during the floral development, indicating that these two genes may play key roles in monoterpene synthesis. Subcellular localization demonstrated that LiDXS and LiDXR are expressed in the chloroplasts. Ectopic expression in transgenic tobacco suggested that the flowers of LiDXS and LiDXR transgenic lines accumulated substantially more diterpene, sclareol, compared to the plants transformed with empty vector. Surprisingly, increased content of the monoterpene, linalool and sesquiterpene, caryophyllene, were detected in the LiDXR transgenic lines, whereas the emission of caryophyllene, increased in one of the LiDXS transgenic tobacco lines, indicating that these two genes play significant roles in the synthesis of floral volatiles in the transgenic plants. These results demonstrate that LiDXR can contribute to monoterpene biosynthesis in Lilium 'Siberia'; however, the role of LiDXS in the biosynthesis of monoterpenes needs further study.

Transcriptome analysis of Crossostephium chinensis provides insight into the molecular basis of salinity stress responses.

Soil salinization is becoming a limitation to the utilization of ornamental plants worldwide. Crossostephium chinensis (Linnaeus) Makino is often cultivated along the southeast coast of China for its desirable ornamental qualities and high salt tolerance. However, little is known about the genomic background of the salt tolerance mechanism in C. chinensis. In the present study, we used Illumina paired-end sequencing to systematically investigate leaf transcriptomes derived from C. chinensis seedlings grown under normal conditions and under salt stress. A total of 105,473,004 bp of reads were assembled into 163,046 unigenes, of which 65,839 (40.38% of the total) and 54,342 (33.32% of the total) were aligned in Swiss-Prot and Nr protein, respectively. A total of 11,331 (6.95%) differentially expressed genes (DEGs) were identified among three comparisons, including 2,239 in 'ST3 vs ST0', 5,880 in 'ST9 vs ST3' and 9,718 in 'ST9 vs ST0', and they were generally classified into 26 Gene Ontology terms and 58 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway terms. Many genes encoding important transcription factors (e.g., WRKY, MYB, and AP2/EREBP) and proteins involved in starch and sucrose metabolism, arginine and proline metabolism, plant hormone signal transduction, amino acid biosynthesis, plant-pathogen interactions and carbohydrate metabolism, among others, were substantially up-regulated under salt stress. These genes represent important candidates for studying the salt-response mechanism and molecular biology of C. chinensis and its relatives. Our findings provide a genomic sequence resource for functional genetic assignments in C. chinensis. These transcriptome datasets will help elucidate the molecular mechanisms responsible for salt-stress tolerance in C. chinensis and facilitate the breeding of new stress-tolerant cultivars for high-saline areas using this valuable genetic resource.