Physiological and transcriptomic profiles reveal key regulatory pathways involved in cold resistance in sunflower seedlings.

During sunflower growth, cold waves often occur and impede plant growth. Therefore, it is crucial to study the underlying mechanism of cold resistance in sunflowers. In this study, physiological analysis revealed that as cold stress increased, the levels of ROS, malondialdehyde, ascorbic acid, and dehydroascorbic acid and the activities of antioxidant enzymes increased. Transcriptomics further identified 10,903 DEGs between any two treatments. Clustering analysis demonstrated that the expression of MYB44a, MYB44b, MYB12, bZIP2 and bZIP4 continuously upregulated under cold stress. Cold stress can induce ROS accumulation, which interacts with hormone signals to activate cold-responsive transcription factors regulating target genes involved in antioxidant defense, secondary metabolite biosynthesis, starch and sucrose metabolism enhancement for improved cold resistance in sunflowers. Additionally, the response of sunflowers to cold stress may be independent of the CBF pathway. These findings enhance our understanding of cold stress resistance in sunflowers and provide a foundation for genetic breeding.

The alleviating effect on the growth, chlorophyll synthesis, and biochemical defense system in sunflowers under cadmium stress achieved through foliar application of humic acid.

BACKGROUND: With the progress of industrialization and urbanization, cadmium (Cd) pollution in farmland is increasingly severe, greatly affecting human health. Sunflowers possess high resistance to Cd stress and great potential for phytoremediation of Cd-contaminated soil. Previous studies have shown that humic acid (HA) effectively mitigates plant damage induced by Cd; however, its alleviating effects on sunflower plants under Cd stress remain largely unknown. RESULTS: We employed four different concentrations of HA (50, 100, 200, and 300 mg L-1) via foliar application to examine their ability to alleviate Cd stress on sunflower plants' growth, chlorophyll synthesis, and biochemical defense system. The results revealed that Cd stress not only reduced plant height, stem diameter, fresh and dry weight, and chlorophyll content in sunflower plants but also altered their chlorophyll fluorescence characteristics compared to the control group. After Cd stress, the photosynthetic structure was damaged and the number of PSII reactive centers per unit changed. Application of 200 mg L-1 HA promotes sunflower growth and increases chlorophyll content. HA significantly enhances antioxidant enzyme activities (SOD, POD, CAT, and APX) and reduces ROS content (O2 -, H2O2 and -OH). Totally, Application of 200 mg L-1 HA had the best effect than other concentrations to alleviate the Cd-induced stress in sunflower plants. CONCLUSIONS: The foliar application of certain HA concentration exhibited the most effective alleviation of Cd-induced stress on sunflower plants. It can enhance the light energy utilization and antioxidant enzyme activities, while reduce ROS contents in sunflower plants. These findings provide a theoretical basis for using HA to mitigate Cd stress in sunflowers.

Development of an Escherichia coli whole cell biocatalyst for the production of hyperoside.

OBJECTIVE: To produce high concentrations of hyperoside from quercetin using recombinant Escherichia coli with in situ regeneration of UDP-galactose. RESULTS: Sucrose synthase from Glycine max (GmSUS) was co-expressed with UDP-glucose epimerase from E. coli (GalE) in E. coli for regenerating UDP-galactose from UDP and sucrose. Glycosyltransferase from Petunia hybrida (PhUGT) was introduced to synthesize hyperoside from quercetin through the regeneration system of UDP-galactose. Co-expressing with molecular chaperones GroEL/ES successfully enhanced the catalytic efficiency of the recombinant strain, which assisted the soluble expression of PhUGT. By using a fed-batch approach, the production of hyperoside reached 863.7 mg L-1 with a corresponding molar conversion of 93.6% and a specific productivity of 72.5 mg L-1 h-1. CONCLUSION: The method described herein for hyperoside production can be widely applied for the synthesis of isorhamnetin-3-O-galactoside, kaempferol-3-O-galactoside and other flavonoids.

GsCLC-c2 from wild soybean confers chloride/salt tolerance to transgenic Arabidopsis and soybean composite plants by regulating anion homeostasis.

The responses of the GsCLC-c2 gene and its promoter to NaCl stress, as well as the Cl- /salt tolerance of GsCLC-c2-transgenic Arabidopsis and overexpressed or RNAi wild soybean hairy root composite plants, were investigated. Results showed that both GsCLC-c2 and its promoter display enhanced induction under salt stress. In the transgenic Arabidopsis WT-GsCLC-c2 and atclc-c-GsCLC-c2 seedlings, the salt-induced growth reduction was markedly ameliorated; plant fresh weight, leaf area, and relative water content (RWC) increased; relative electrolytic leakage (REL), and malondialdehyde (MDA) content in shoots decreased significantly. In addition, accumulation of Cl- and K+ , especially Cl- , increased markedly in roots to minimize Cl- transport to shoots and maintain higher and lower Cl- /NO3 - ratios in roots and shoots, respectively. When compared to GsCLC-c2-RNAi wild soybean composite plants under salt stress, clear advantages, such as growth appearance, plant height, and leaf area, were displayed by GsCLC-c2-overexpressing composite plants. Moreover, their REL values in roots and leaves declined significantly. The accumulation of absorbed Cl- and Na+ in the roots increased, as the transportation to the stems and leaves decreased, the NO3 - content in roots, stems, and leaves significantly increased, and the changes in K+ contents were small, which resulted in the maintenance of a low Cl- /NO3 - ratio in all plant parts and low Na+ /K+ ratio in stems and leaves. Taken together, these results highlight the role of GsCLC-c2 in regulating anionic homeostasis in NaCl-stressed transgenic Arabidopsis and soybean composite plants to maintain lower Cl- /NO3 - ratios in shoots, thus conferring enhanced Cl- /salt tolerance.

Engineering a heterologous synthetic pathway in Escherichia coli for efficient production of biotin.

OBJECTIVE: To enhance biotin production in Escherichia coli by engineering a heterologous biotin synthetic pathway. RESULTS: Biotin operon genes from Pseudomonas putida, which consisted of a bioBFHCD cluster and a bioA gene, was engineered into Escherichia coli for biotin production. The introduction of bioW gene from Bacillus subtilis, encoding pimeloyl-CoA synthetase and sam2 gene from Saccharomyces cerevisiae, encoding S-adenosyl-L-methionine (SAM) synthetase contributed to the heterologous production of biotin in recombinant E. coli. Furthermore, biotin production was efficiently enhanced by optimization of the fermentation compositions, especially pimelic acid and L-methionine, the precursor related to the pimeloyl-CoA and SAM synthesis, respectively. The combination of overexpression of the heterologous biotin operon genes and enhanced supply of key intermediate pimeloyl-CoA and SAM increased biotin production in E. coli by more than 121-fold. With bioprocess engineering efforts, biotin was produced at a final titer of 92.6 mg/L in a shake flask and 208.7 mg/L in a fed-batch fermenter. CONCLUSION: Through introduction of heterologous biotin synthetic pathway, increasing the supply of precursor pimeloyl-CoA and cofactor SAM can significantly enhance biotin production in E. coli.

Identification and functional characterization of the chloride channel gene, GsCLC-c2 from wild soybean.

BACKGROUND: The anionic toxicity of plants under salt stress is mainly caused by chloride (Cl-). Thus Cl- influx, transport and their regulatory mechanisms should be one of the most important aspects of plant salt tolerance studies, but are often sidelined by the focus on sodium (Na+) toxicity and its associated adaptations. Plant chloride channels (CLCs) are transport proteins for anions including Cl- and nitrate (NO3-), and are critical for nutrition uptake and transport, adjustment of cellular turgor, stomatal movement, signal transduction, and Cl- and NO3- homeostasis under salt stress. RESULTS: Among the eight soybean CLC genes, the tonoplast-localized c2 has uniquely different transcriptional patterns between cultivated soybean N23674 and wild soybean BB52. Using soybean hairy root transformation, we found that GsCLC-c2 over-expression contributed to Cl- and NO3- homeostasis, and therefore conferred salt tolerance, through increasing the accumulation of Cl- in the roots, thereby reducing their transportation to the shoots where most of the cellular damages occur. Also, by keeping relatively high levels of NO3- in the aerial part of the plant, GsCLC-c2 could reduce the Cl-/NO3- ratio. Wild type GsCLC-c2, but not its mutants (S184P, E227V and E294G) with mutations in the conserved domains, is able to complement Saccharomyces cerevisiae △gef1 Cl- sensitive phenotype. Using two-electrode voltage clamp on Xenopus laevis oocytes injected with GsCLC-c2 cRNA, we found that GsCLC-c2 transports both Cl- and NO3- with slightly different affinity, and the affinity toward Cl- was pH-independent. CONCLUSION: This study revealed that the expression of GsCLC-c2 is induced by NaCl-stress in the root of wild soybean. The tonoplast localized GsCLC-c2 transports Cl- with a higher affinity than NO3- in a pH-independent fashion. GsCLC-c2 probably alleviates salt stress in planta through the sequestration of excess Cl- into the vacuoles of root cells and thus preventing Cl- from entering the shoots where it could result in cellular damages.

Recent Advances in the Development of Water Oxidation Electrocatalysts at Mild pH.

Developing anodic oxygen evolution reaction (OER) electrocatalysts with high catalytic activities is of great importance for effective water splitting. Compared with the water-oxidation electrocatalysts that are commonly utilized in alkaline conditions, the ones operating efficiently under neutral or near neutral conditions are more environmentally friendly with less corrosion issues. This review starts with a brief introduction of OER, the importance of OER in mild-pH media, as well as the fundamentals and performance parameters of OER electrocatalysts. Then, recent progress of the rational design of electrocatalysts for OER in mild-pH conditions is discussed. The chemical structures or components, synthetic approaches, and catalytic performances of the OER catalysts will be reviewed. Some interesting insights into the catalytic mechanism are also included and discussed. It concludes with a brief outlook on the possible remaining challenges and future trends of neutral or near-neutral OER electrocatalysts. It hopefully provides the readers with a distinct perspective of the history, present, and future of OER electrocatalysts at mild conditions.

Trends in the consecutive days of temperature and precipitation extremes in China during 1961-2015.

BACKGROUND AND AIMS: Consecutive climatic extremes have more intense impacts on natural ecosystems and human activities than occasional events. There were many studies about the frequency or intensity of extreme weather events, but few focused on the consecutiveness or continuousness of climatic extremes. We analyzed the temporal and spatial distributions and tendencies in the consecutive temperature and precipitation extremes in China during 1961-2015. METHODS: Daily temperature and precipitation data at 1867 meteorological stations over China was used and four consecutive indices of climate extremes, i.e. cold spell duration indicator (CSDI), warm spell duration indicator (WSDI), consecutive dry days (CDD) and consecutive wet days (CWD), were calculated by RClimDex 1.0. Linear trends in the time series of consecutive days of temperature and precipitation extremes were examined and their statistical significance was evaluated using Mann-Kendall test. RESULTS AND DISCUSSION: There were obvious differences in the spatial distributions of consecutive days of climate extremes in China. During 1961-2015, CSDI and CWD decreased significantly at rates of 0.9 and 0.1 days per decade respectively, while WSDI increased significantly at rate of 0.8 days per decade in China. Spatially, CSDI decreased at rates of 0-3.0 days per decade in almost all parts of China, and WSDI increased at rates of 0-2.0 days per decade in most parts of China. The spatial trends of CDD and CWD were significant only in several regions of China. CSDI and WSDI had higher percent changes than those of CDD and CWD. Changes in the CSDI and WSDI were associated with large-scale oceanic and atmospheric circulation oscillations, such as Atlantic Multidecadal Oscillation (AMO), El Niño/Southern Oscillation (ENSO) and Pacific Decadal Oscillation (PDO). With global warming, there will be fewer cold extremes, more frequent hot extremes and precipitation extremes. CONCLUSIONS: Given the increases in the frequency and intensity of some consecutive climatic extremes and an increasing physical exposure and socio-economic vulnerability to such extremes in China, more strategies and capacities of mitigation and adaptation to consecutive climatic extremes are essential for the local government and climate-sensitive sectors.

Identification of nuclear low-copy genes and their phylogenetic utility in rosids.

By far, the interordinal relationships in rosids remain poorly resolved. Previous studies based on chloroplast, mitochondrial, and nuclear DNA has produced conflicting phylogenetic resolutions that has become a widely concerned problem in recent phylogenetic studies. Here, a total of 96 single-copy nuclear gene loci were identified from the KOG (eukaryotic orthologous groups) database, most of which were first used for phylogenetic analysis of angiosperms. The orthologous sequence datasets from completely sequenced genomes of rosids were assembled for the resolution of the position of the COM (Celastrales-Oxalidales-Malpighiales) clade in rosids. Our analysis revealed strong and consistent support for CM topology (the COM clade as sister to the malvids). Our results will contribute to further exploring the underlying cause of conflict between chloroplast, mitochondrial, and nuclear data. In addition, our study identified a few novel nuclear molecular markers with potential to investigate the deep phylogenetic relationship of plants or other eukaryotic taxonomical groups.

Genome-wide identification and expression analysis of the Dof gene family reveals their involvement in hormone response and abiotic stresses in sunflower (Helianthus annuus L.).

DNA binding with one finger (Dof), plant-specific zinc finger transcription factors, can participate in various physiological and biochemical processes during the life of plants. As one of the most important oil crops in the world, sunflower (Helianthus annuus L.) has significant economic and ornamental value. However, a systematic analysis of H. annuus Dof (HaDof) members and their functions has not been extensively conducted. In this study, we identified 50 HaDof genes that are unevenly distributed on 17 chromosomes of sunflower. We present a comprehensive overview of the HaDof genes, including their chromosome locations, phylogenetic analysis, and expression profile characterization. Phylogenetic analysis classified the 366 Dof members identified from 11 species into four groups (further subdivided into nine subfamilies). Segmental duplications are predominantly contributed to the expansion of sunflower Dof genes, and all segmental duplicate gene pairs are under purifying selection due to strong evolutionary constraints. Furthermore, we observed differential expression patterns for HaDof genes in normal tissues as well as under hormone treatment or abiotic stress conditions by analyzing RNA-seq data from previous studies and RT-qPCR data in our current study. The expression of HaDof04 and HaDof43 were not detected in any samples, which implied that they may be gradually undergoing pseudogenization process. Some HaDof genes, such as HaDof25 and HaDof30, showed responsiveness to exogenous plant hormones, such as kinetin, brassinosteroid, auxin or strigolactone, while others like HaDof15 and HaDof35 may participate in abiotic stress resistance of sunflower seedling. Our study represents the initial step towards understanding the phylogeny and expression characterization of sunflower Dof family genes, which may provide valuable reference information for functional studies on hormone response, abiotic stress resistance, and molecular breeding in sunflower and other species.

Time and power dependence of laser-induced photodamage on human sperm revealed by longitudinal rolling measurement using optical tweezers.

Lasers are widely applied in assisted reproductive technologies, including sperm fixation, sperm selection and intracytoplasmic sperm injections, to reduce procedure time and improve consistency and reproducibility. However, quantitative studies on laser-induced photodamage of sperm are lacking. In this study, we demonstrated that, by using optical tweezers, the kinematic parameters of freely swimming sperm are correlated with the frequency as well as the percentage of pausing duration of longitudinal rolling of the same sperm head in the optical trap. Furthermore, by trapping individual sperm cells using 1064-nm optical tweezers, we quantitatively characterized the time-dependence of longitudinal rolling frequency and percentage of pausing duration of sperm under different laser powers. Our study revealed that, as trapping time and the laser power time increase, the longitudinal rolling frequency of the optically trapped sperm decreases with an increasing percentage of pausing duration, which characterizes the effect of laser power and duration on the photodamage of individual sperm cells. Our study provides experimental basis for the optimization of laser application in assisted reproductive technology, which may reduce the photodamage-induced biosafety risk in the future.

A chromosome-scale genome of Peucedanum praeruptorum provide insights into Apioideae evolution and medicinal ingredient biosynthesis.

Peucedanum praeruptorum Dunn, a traditional Chinese medicine rich in coumarin, belongs to the Apiaceae family. A high-quality assembled genome of P. praeruptorum is lacking, which has posed obstacles to functional identification and molecular evolution studies of genes associated with coumarin production. Here, a chromosome-scale reference genome of P. praeruptorum, an important medicinal and aromatic plant, was first sequenced and assembled using Oxford Nanopore Technologies and Hi-C sequencing. The final assembled genome size was 1.83 Gb, with a contig N50 of 11.12 Mb. The entire BUSCO evaluation and second-generation read comparability rates were 96.0 % and 99.31 %, respectively. Furthermore, 99.91 % of the genome was anchored to 11 pseudochromosomes. The comparative genomic study revealed the presence of 18,593 orthogroups, which included 476 species-specific orthogroups and 1211 expanded gene families. Two whole-genome duplication (WGD) events and one whole-genome triplication (WGT) event occurred in P. praeruptorum. In addition to the γ-WGT shared by core eudicots or most eudicots, the first WGD was shared by Apiales, while the most recent WGD was unique to Apiaceae. Our study demonstrated that WGD events that occurred in Apioideae highlighted the important role of tandem duplication in the biosynthesis of coumarins and terpenes in P. praeruptorum. Additionally, the expansion of the cytochrome P450 monooxygenase, O-methyltransferase, ATP-binding cassette (ABC) transporter, and terpene synthase families may be associated with the abundance of coumarins and terpenoids. Moreover, we identified >170 UDP-glucosyltransferase members that may be involved in the glycosylation post-modification of coumarins. Significant gene expansion was observed in the ABCG, ABCB, and ABCC subgroups of the ABC transporter family, potentially facilitating the transmembrane transport of coumarins after bolting. The P. praeruptorum genome provides valuable insights into the machinery of coumarin biosynthesis and enhances our understanding of Apiaceae evolution.

Lead Drug Discover Strategies from Natural Medicines Based on Network Pharmacology.

The need for therapeutics to overcome development of existing diseases research to discover new lead agents. In the face of public health challenges worldwide, natural medicines play a pivotal role in innovative lead drug discovery. Network pharmacology can easily construct complicated poly-pharmacology network based on lead compound, biological function, and bioactive target proteins, which meets the overall feature of natural medicines, and enable to elucidate the action mechanism at molecule-protein level with systematic view. In this work, we first summarized the recent progress delineating lead drug development and its interaction with natural medicines. Second, we focused on the relationship between natural medicines and network pharmacology. Additionally, we discussed current issues and potential prospects for the lead drug discover from natural medicines by network pharmacology. Further investigations should be focus on relevant structural analysis for biological experiment, also the dynamic and quantitative network development. In summary, it is a rational approach for innovative lead drug discovery, and with the development of structure and biology research, this approach makes it a very powerful method for the lead molecules in a high-throughput manner from a comprehensive and powerful special multi-compound to target protein/disease poly pharmacology network.

Rational engineering of a metalloprotease to enhance thermostability and activity.

The rational design of enzymes with enhanced thermostability is efficient. Solvent-tolerant metalloprotease from Pseudomonas aeruginosa PT121 presents high Z-aspartame (Z-APM) synthesis activity, but insufficient thermostability. In this study, we enhanced enzyme thermostability using a rational strategy. Molecular dynamics (MD) simulation was applied to rapidly identify that the D28 and D116 mutations are likely to exhibit increased thermostability, and experimentation verified that the D28N and D116N mutants were more stable than the wild-type (WT) enzyme. In particular, the Tm of the D28N and D116N mutants increased by 6.1 °C and 9.2 °C, respectively, compared with that of the WT enzyme. The half-lives of D28N and D116N at 60 °C were 1.07- and 1.8-fold higher than that of the WT, respectively. Z-APM synthetic activities of the mutants were also improved. The potential mechanism of thermostability enhancement rationalized using MD simulation indicated that increased hydrogen bond interactions and a regional hydration shell were mostly responsible for the thermostability enhancement. Our strategy could be a reference for enzyme engineering, and our mutants offer considerable value in industrial applications.

Label-Free Quantitative Proteomics Unravel the Impacts of Salt Stress on Dendrobium huoshanense.

Salt stress is a constraint on crop growth and productivity. When exposed to high salt stress, metabolic abnormalities that disrupt reactive oxygen species (ROS) homeostasis result in massive oxygen radical deposition. Dendrobium huoshanense is a perennial orchid herb that thrives in semi-shade conditions. Although lots of studies have been undertaken on abiotic stresses (high temperature, chilling, drought, etc.) of model plants, few studies were reported on the mechanism of salt stress in D. huoshanense. Using a label-free protein quantification method, a total of 2,002 differential expressed proteins were identified in D. huoshanense. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment indicated that proteins involved in vitamin B6 metabolism, photosynthesis, spliceosome, arginine biosynthesis, oxidative phosphorylation, and MAPK signaling were considerably enriched. Remarkably, six malate dehydrogenases (MDHs) were identified from deferentially expressed proteins. (NAD+)-dependent MDH may directly participate in the biosynthesis of malate in the nocturnal crassulacean acid metabolism (CAM) pathway. Additionally, peroxidases such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), as well as antioxidant enzymes involved in glutathione biosynthesis and some vitamins biosynthesis were also identified. Taken together, these results provide a solid foundation for the investigation of the mechanism of salt stress in Dendrobium spp.

In-depth analysis of genomes and functional genomics of orchid using cutting-edge high-throughput sequencing.

High-throughput sequencing technology has been facilitated the development of new methodologies and approaches for studying the origin and evolution of plant genomes and subgenomes, population domestication, and functional genomics. Orchids have tens of thousands of members in nature. Many of them have promising application potential in the extension and conservation of the ecological chain, the horticultural use of ornamental blossoms, and the utilization of botanical medicines. However, a large-scale gene knockout mutant library and a sophisticated genetic transformation system are still lacking in the improvement of orchid germplasm resources. New gene editing tools, such as the favored CRISPR-Cas9 or some base editors, have not yet been widely applied in orchids. In addition to a large variety of orchid cultivars, the high-precision, high-throughput genome sequencing technology is also required for the mining of trait-related functional genes. Nowadays, the focus of orchid genomics research has been directed to the origin and classification of species, genome evolution and deletion, gene duplication and chromosomal polyploidy, and flower morphogenesis-related regulation. Here, the progressing achieved in orchid molecular biology and genomics over the past few decades have been discussed, including the evolution of genome size and polyploidization. The frequent incorporation of LTR retrotransposons play important role in the expansion and structural variation of the orchid genome. The large-scale gene duplication event of the nuclear genome generated plenty of recently tandem duplicated genes, which drove the evolution and functional divergency of new genes. The evolution and loss of the plastid genome, which mostly affected genes related to photosynthesis and autotrophy, demonstrated that orchids have experienced more separate transitions to heterotrophy than any other terrestrial plant. Moreover, large-scale resequencing provide useful SNP markers for constructing genetic maps, which will facilitate the breeding of novel orchid varieties. The significance of high-throughput sequencing and gene editing technologies in the identification and molecular breeding of the trait-related genes in orchids provides us with a representative trait-improving gene as well as some mechanisms worthy of further investigation. In addition, gene editing has promise for the improvement of orchid genetic transformation and the investigation of gene function. This knowledge may provide a scientific reference and theoretical basis for orchid genome studies.

The combination of RNA-seq transcriptomics and data-independent acquisition proteomics reveals the mechanisms underlying enhanced salt tolerance by the ZmPDI gene in Zoysia matrella [L.] Merr.

Zoysia matrella [L.] Merr. is one of the three most economically important Zoysia species due to its strong salt tolerance and wide application. However, the molecular mechanisms regulating salt tolerance in Z. matrella remain unknown. The protein disulfide isomerase ZmPDI of Z. matrella was obtained by salt stress screening with yeast cells, and its expression was significantly upregulated after salt stress. Based on the obtained ZmPDI overexpression transgenic Z. matrella plants, we carried out salt tolerance identification and found that ZmPDI can significantly enhance the salt tolerance of Z. matrella. Root samples of OX-ZmPDI transgenic and wild-type plants were collected at 0 and 24 h after salt treatments for RNA-seq and data-independent acquisition (DIA) proteome sequencing. Combined analysis of the transcriptome and proteome revealed that ZmPDI may enhance the salt tolerance of Z. matrella by regulating TUBB2, PXG4, PLDα2, PFK4, and 4CL1. This research presents the molecular regulatory mechanism of the ZmPDI gene in Z. matrella for resistance to salt stress and facilitates the use of molecular breeding to improve the salt tolerance of grasses.

Genome-wide meta-analysis of QTL for morphological related traits of flag leaf in bread wheat.

Flag leaf is an important organ for photosynthesis of wheat plants, and a key factor affecting wheat yield. In this study, quantitative trait loci (QTL) for flag leaf morphological traits in wheat reported since 2010 were collected to investigate the genetic mechanism of these traits. Integration of 304 QTLs from various mapping populations into a high-density consensus map composed of various types of molecular markers as well as QTL meta-analysis discovered 55 meta-QTLs (MQTL) controlling morphological traits of flag leaves, of which 10 MQTLs were confirmed by GWAS. Four high-confidence MQTLs (MQTL-1, MQTL-11, MQTL-13, and MQTL-52) were screened out from 55 MQTLs, with an average confidence interval of 0.82 cM and a physical distance of 9.4 Mb, according to the definition of hcMQTL. Ten wheat orthologs from rice (7) and Arabidopsis (3) that regulated leaf angle, development and morphogenesis traits were identified in the hcMQTL region using comparative genomics, and were speculated to be potential candidate genes regulating flag leaf morphological traits in wheat. The results from this study provides valuable information for fine mapping and molecular markers assisted selection to improve morphological characters in wheat flag leaf.

Selection of Suitable Reference Genes for Gene Expression Normalization Studies in Dendrobium huoshanense.

Dendrobium huoshanense is a kind of precious herb with important medicinal and edible value in China, which is widely used in traditional Chinese medicine for various diseases. Recent studies have paid close attention to the genetic expression of the biosynthetic pathway of the main active components (polysaccharides, alkaloids, and flavonoids), and real-time polymerase chain reaction (qPCR) is one of the most widely used methods for doing so. However, so far, no reference gene selections have been reported in D. huoshanense. In this study, 15 reference gene candidates (GAPDH, eIF, EF-1α, PP2A, UBCE, RPL5, TBP, APT1, MDH, PTBP3, PEPC, CYP71, NCBP2, TIP41, and F-box) were selected and evaluated for their expression stability in D. huoshanense under various experimental conditions, including in different tissues (root, stem, and leaf), abiotic stresses (oxidative, drought, cold, and UV), and hormone treatment (methyl jasmonate) using three statistical programs (geNorm, NormFinder, and BestKeeper). Then, the RefFinder program was employed to comprehensively validate the stability of the selected reference genes. Finally, the expression profiles of the CESA and GMPP genes were further analyzed, and these results indicated that TBP, NCBP2, and CYP71 were the top three most stable reference genes after comprehensive comparison, which could be used as stable reference genes for normalizing the genes expression in D. huoshanense. This study described here provides the first data regarding on reference gene selection in D. huoshanense, which will be extremely beneficial for future research on the gene expression normalization in D. huoshanense.

Metabolic Engineering of Escherichia coli for Hyperoside Biosynthesis.

Hyperoside (quercetin 3-O-galactoside) exhibits many biological functions, along with higher bioactivities than quercetin. In this study, three UDP-dependent glycosyltransferases (UGTs) were screened for efficient hyperoside synthesis from quercetin. The highest hyperoside production of 58.5 mg·L-1 was obtained in a recombinant Escherichia coli co-expressing UGT from Petunia hybrida (PhUGT) and UDP-glucose epimerase (GalE, a key enzyme catalyzing the conversion of UDP-glucose to UDP-galactose) from E. coli. When additional enzymes (phosphoglucomutase (Pgm) and UDP-glucose pyrophosphorylase (GalU)) were introduced into the recombinant E. coli, the increased flux toward UDP-glucose synthesis led to enhanced UDP-galactose-derived hyperoside synthesis. The efficiency of the recombinant strain was further improved by increasing the copy number of the PhUGT, which is a limiting step in the bioconversion. Through the optimization of the fermentation conditions, the production of hyperoside increased from 245.6 to 411.2 mg·L-1. The production was also conducted using a substrate-fed batch fermentation, and the maximal hyperoside production was 831.6 mg·L-1, with a molar conversion ratio of 90.2% and a specific productivity of 27.7 mg·L-1·h-1 after 30 h of fermentation. The efficient hyperoside synthesis pathway described here can be used widely for the glycosylation of other flavonoids and bioactive substances.