Integration of Metabolome and Transcriptome Reveals the Relationship of Benzenoid-Phenylpropanoid Pigment and Aroma in Purple Tea Flowers.

Tea (Camellia sinensis) flowers are normally white, even though the leaves could be purple. We previously discovered a specific variety with purple leaves and flowers. In the face of such a phenomenon, researchers usually focus on the mechanism of color formation but ignore the change of aroma. The purple tea flowers contain more anthocyanins, which belong to flavonoids. Meanwhile, phenylalanine (Phe), derived from the shikimate pathway, is a precursor for both flavonoids and volatile benzenoid-phenylpropanoids (BPs). Thus, it is not clear whether the BP aroma was attenuated for the appearance of purple color. In this study, we integrated metabolome and transcriptome of petals of two tea varieties, namely, Zijuan (ZJ) with white flowers and Baitang (BT) with purple flowers, to reveal the relationship between color (anthocyanins) and aroma (volatile BPs). The results indicated that in purple petals, the upstream shikimate pathway promoted for 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) was elevated. Among the increased anthocyanins, delphinidin-3-O-glucoside (DpG) was extremely higher; volatile BPs, including benzyl aldehyde, benzyl alcohol, acetophenone (AP), 1-phenylethanol, and 2-phenylethanol, were also enhanced, and AP was largely elevated. The structural genes related to the biosynthesis of volatile BPs were induced, while the whole flavonoid biosynthesis pathway was downregulated, except for the genes flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H), which were highly expressed to shift the carbon flux to delphinidin, which was then conjugated to glucoside by increased bronze-1 (BZ1) (UDP-glucose: flavonoid 3-O-glucosyltransferase) to form DpG. Transcription factors (TFs) highly related to AP and DpG were selected to investigate their correlation with the differentially expressed structural genes. TFs, such as MYB, AP2/ERF, bZIP, TCP, and GATA, were dramatically expressed and focused on the regulation of genes in the upstream synthesis of Phe (DAHPS; arogenate dehydratase/prephenate dehydratase) and the synthesis of AP (phenylacetaldehyde reductase; short-chain dehydrogenase/reductase), Dp (F3'H; F3'5'H), and DpG (BZ1), but inhibited the formation of flavones (flavonol synthase) and catechins (leucoanthocyanidin reductase). These results discovered an unexpected promotion of volatile BPs in purple tea flowers and extended our understanding of the relationship between the BP-type color and aroma in the tea plant.

Peripheral Artery Disease and Risk of Fibrosis Deterioration in Nonalcoholic Fatty Liver Disease: A Prospective Investigation.

OBJECTIVE: Liver fibrosis is an important predictor of mortality in nonalcoholic fatty liver disease (NAFLD). Peripheral artery disease (PAD) and liver fibrosis share many common metabolic dysfunctions. We aimed to explore the association between PAD and risk of fibrosis deterioration in NAFLD patients. METHODS: The study recruited 1,610 NAFLD patients aged ≥ 40 years from a well-defined community at baseline in 2010 and followed up between August 2014 and May 2015. Fibrosis deterioration was defined as the NAFLD fibrosis score (NFS) status increased to a higher category at the follow-up visit. PAD was defined as an ankle-brachial index of < 0.90 or > 1.40. RESULTS: During an average of 4.3 years' follow-up, 618 patients progressed to a higher NFS category. PAD was associated with 92% increased risk of fibrosis deterioration [multivariable-adjusted odds ratio ( OR): 1.92, 95% confidence interval ( CI): 1.24, 2.98]. When stratified by baseline NFS status, the OR for progression from low to intermediate or high NFS was 1.74 (95% CI: 1.02, 3.00), and progression from intermediate to high NFS was 2.24 (95% CI: 1.05, 4.80). There was a significant interaction between PAD and insulin resistance (IR) on fibrosis deterioration ( P for interaction = 0.03). As compared with non-PAD and non-IR, the coexistence of PAD and IR was associated with a 3.85-fold (95% CI: 2.06, 7.18) increased risk of fibrosis deterioration. CONCLUSION: PAD is associated with an increased risk of fibrosis deterioration in NAFLD patients, especially in those with IR. The coexistence of PAD and IR may impose an interactive effect on the risk of fibrosis deterioration.

Transcriptome responses to elevated CO2 level and Wolbachia-infection stress in Hylyphantes graminicola (Araneae: Linyphiidae).

Hylyphantes graminicola is a resident spider species found in maize and cotton fields and is an important biological control agent of various pests. Previous studies have demonstrated that stress from elevated CO2 and Wolbachia infection can strongly affect spider species. Thus, based on CO2 levels (400 ppm, current atmospheric CO2 concentration and 800 ppm, high CO2 concentration) and Wolbachia status (Wolbachia-infected, W+ and Wolbachia-uninfected, W- ), we divided H. graminicola individuals into four treatment groups: W- 400 ppm, W- 800 ppm, W+ 400 ppm, and W+ 800 ppm. To investigate the effects of elevated CO2 levels (W- 400 vs W- 800), Wolbachia infection (W- 400 vs W+ 400), and the interactions between these two factors (W- 400 vs W+ 800), high-throughput transcriptome sequencing was employed to characterize the de novo transcriptome of the spiders and identify stress-related differentially expressed genes (DEGs). De novo assembly of complementary DNA sequences generated 86 688 unigenes, 23 938 of which were annotated in public databases. A total of 84, 21, and 157 DEGs were found among W- 400 vs W- 800, W- 400 vs W+ 400, and W- 400 vs W+ 800, respectively. Functional enrichment analysis revealed that metabolic processes, signaling, and catalytic activity were significantly affected by elevated CO2 levels and Wolbachia infection. Our findings suggest that the impact of elevated CO2 levels and Wolbachia infection on the H. graminicola transcriptome was, to a large extent, on genes involved in metabolic processes. This study is the first description of transcriptome changes in response to elevated CO2 levels and Wolbachia infection in spiders.

Convergence between a mosquito-eating predator's natural diet and its prey-choice behaviour.

On the basis of 1115 records of Evarcha culicivora feeding in the field, we can characterize this East African jumping spider (Salticidae) as being distinctively stenophagic. We can also, on the basis of laboratory prey-choice experiments, characterize E. culicivora as having a specialized prey-classification system and a hierarchy of innate preferences for various categories of mosquitoes and other arthropods. Prey from the field belonged to 10 arthropod orders, but 94.5% of the prey records were dipterans. Mosquitoes were the dominant prey (80.2% of the records), with the majority (82.9%) of the mosquitoes being females, and thereafter midges were the most common prey (9.2% of the records). Preference profiles that were determined from experiments showed strong convergence with natural diet in some, but not all, instances. In experiments, E. culicivora adults appeared to distinguish between six prey categories and juveniles between seven, with blood-carrying anopheline female mosquitoes being ranked highest in preference. For adults, this was followed by blood-carrying culicine female mosquitoes and then anopheline female mosquitoes not carrying blood, but these two preferences were reversed for juveniles. Moreover, for juveniles, but not for adults, anopheline male mosquitoes seem to be a distinct prey category ranked in preference after blood-carrying culicine females and, for both adults and juveniles, preference for midges is evident when the alternatives are not mosquitoes. These findings illustrate the importance of going beyond simply specifying preferred prey categories when characterizing predators as 'specialized' and a need to make clear conceptual distinctions between a predator's natural diet, the prey categories that are relevant to the predator, and the predator's prey-choicebehaviour.

Directed evolution of Streptomyces clavuligerus deacetoxycephalosporin C synthase for enhancement of penicillin G expansion.

The deacetoxycephalosporin C synthase from Streptomyces clavuligerus was directly modified for enhancement of penicillin G expansion into phenylacetyl-7-aminodeacetoxycephalosporanic acid, an important intermediate in the industrial manufacture of cephalosporin antibiotics. Nine new mutants, mutants M73T, T91A, A106T, C155Y, Y184H, M188V, M188I, H244Q, and L277Q with 1.4- to 5.7-fold increases in the kcat/Km ratio, were obtained by screening 6,364 clones after error-prone PCR-based random mutagenesis. Subsequently, DNA shuffling was carried out to screen possible combinations of substitutions, including previous point mutations. One quaternary mutant, the C155Y/Y184H/V275I/C281Y mutant, which had a kcat/Km ratio that was 41-fold higher was found after 10,572 clones were assayed. The distinct mutants obtained using different mutagenesis methods demonstrated the complementarity of the techniques. Interestingly, most of the mutated residues that result in enhanced activities are located within or near the unique small barrel subdomain, suggesting that manipulation of this subdomain may be a constructive strategy for improvement of penicillin expansion. Several mutations had very distinct effects on expansion of penicillins N and G, perhaps due to different penicillin-interacting modes within the enzyme. Thus, the present study provided not only promising enzymes for cephalosporin biosynthesis but also a large number of mutants, which provided new insights into the structure-function relationship of the protein that should lead to further rational engineering.

Family shuffling of expandase genes to enhance substrate specificity for penicillin G.

Deacetoxycephalosporin C synthase (expandase) from Streptomyces clavuligerus, encoded by cefE, is an important industrial enzyme for the production of 7-aminodeacetoxycephalosporanic acid from penicillin G. To improve the substrate specificity for penicillin G, eight cefE-homologous genes were directly evolved by using the DNA shuffling technique. After the first round of shuffling and screening, using an Escherichia coli ESS bioassay, four chimeras with higher activity were subjected to a second round. Subsequently, 20 clones were found with significantly enhanced activity. The kinetic parameters of two isolates that lack substrate inhibition showed 8.5- and 118-fold increases in the k(cat)/K(m) ratio compared to the S. clavuligerus expandase. The evolved enzyme with the 118-fold increase is the most active obtained to date anywhere. Our shuffling results also indicate the remarkable plasticity of the expandase, suggesting that more-active chimeras might be achievable with further rounds.