Functional characterization of geranyl/farnesyl diphosphate synthase in Wurfbainia villosa and Wurfbainia longiligularis.

Wurfbainia villosa and Wurfbainia longiligularis are the two primary plant sources of Fructus Amomi, a traditional Chinese medicine. Both plants are rich in volatile terpenoids, including monoterpenes and sesquiterpenes, which are the primary medicinal components of Fructus Amomi. The trans-isopentenyl diphosphate synthase (TIDS) gene family plays a key part in determining terpenoid diversity and accumulation. However, the TIDS gene family have not been identified in W. villosa and W. longiligularis. This study identified thirteen TIDS genes in W. villosa and eleven TIDS genes in W. longiligularis, which may have expanded through segmental replication events. Based on phylogenetic analysis and expression levels, eight candidate WvTIDSs and five WlTIDSs were selected for cloning. Functional characterization in vitro demonstrated that four homologous geranyl diphosphate synthases (GPPSs) (WvGPPS1, WvGPPS2, WlGPPS1, WlGPPS2) and two geranylgeranyl diphosphate synthases (GGPPSs) (WvGGPPS and WlGGPPS) were responsible for catalyzing the biosynthesis of geranyl diphosphate (GPP), whereas two farnesyl diphosphate synthases (FPPSs) (WvFPPS and WlFPPS) catalysed the biosynthesis of the farnesyl diphosphate (FPP). A comparison of six proteins with identified GPPS functions showed that WvGGPPS and WlGGPPS exhibited the highest activity levels. These findings indicate that homologous GPPS and GGPPS together promote the biosynthesis of GPP in W. villosa and W. longiligularis, thus providing sufficient precursors for the synthesis of monoterpenes and providing key genetic elements for Fructus Amomi variety improvement and molecular breeding.

Substrate and functional characterization of the lysine acetyltransferase MsKat and deacetylase MsCobB in Mycobacterium smegmatis.

Tuberculosis (TB) is a serious cause of infectious death worldwide. Recent studies have reported that about 30% of the Mtb proteome was modified post-translationally, indicating that their functions are essential for drug resistance, mycobacterial survival, and pathogenicity. Among them, lysine acetylation, reversibly regulated by acetyltransferase and deacetylase, has important roles involved in energy metabolism, cellular adaptation, and protein interactions. However, the substrate and biological functions of these two important regulatory enzymes remain unclear. Herein, we utilized the non-pathogenic M. smegmatis strain as a model and systematically investigated the dynamic proteome changes in response to the overexpressing of MsKat/MsCobB in mycobacteria. A total of 4179 proteins and 1236 acetylated sites were identified in our data. Further analysis of the dynamic changes involved in proteome and acetylome showed that MsKat/MsCobB played a regulatory role in various metabolic pathways and nucleic acid processes. After that, the quantitative mass spectrometric method was utilized and proved that the AMP-dependent synthetase, Citrate synthase, ATP-dependent specificity component of the Clp protease, and ATP-dependent DNA/RNA helicases were identified to be the substrates of MsKat. Overall, our study provided an important resource underlying the substrates and functions of the acetylation regulatory enzymes in mycobacteria. SIGNIFICANCE: In this study, we systematically analyzed the dynamic molecular changes in response to the MsKat/MsCobB overexpression in mycobacteria at proteome and lysine acetylation level by using a TMT-based quantitative proteomic approach. Pathways related with glycolysis, degradation of branched chain amino acids, phosphotransferase system were affected after disturbance of the two regulates enzymes involved in lysine acetylation. We also proved that AMP-dependent synthetase Clp protease, ATP-dependent DNA/RNA helicases and citrate synthase was the substrate of MsKat according to our proteomic data and biological validation. Together, our study underlined the substrates and functions of the acetylation regulatory enzymes in mycobacteria.

Comparing genomes of Fructus Amomi-producing species reveals genetic basis of volatile terpenoid divergence.

Wurfbainia longiligularis and Wurfbainia villosa are both rich in volatile terpenoids and are 2 primary plant sources of Fructus Amomi used for curing gastrointestinal diseases. Metabolomic profiling has demonstrated that bornyl diphosphate (BPP)-related terpenoids are more abundant in the W. villosa seeds and have a wider tissue distribution in W. longiligularis. To explore the genetic mechanisms underlying the volatile terpenoid divergence, a high-quality chromosome-level genome of W. longiligularis (2.29 Gb, contig N50 of 80.39 Mb) was assembled. Functional characterization of 17 terpene synthases (WlTPSs) revealed that WlBPPS, along with WlTPS 24/26/28 with bornyl diphosphate synthase (BPPS) activity, contributes to the wider tissue distribution of BPP-related terpenoids in W. longiligularis compared to W. villosa. Furthermore, transgenic Nicotiana tabacum showed that the GCN4-motif element positively regulates seed expression of WvBPPS and thus promotes the enrichment of BPP-related terpenoids in W. villosa seeds. Systematic identification and analysis of candidate TPS in 29 monocot plants from 16 families indicated that substantial expansion of TPS-a and TPS-b subfamily genes in Zingiberaceae may have driven increased diversity and production of volatile terpenoids. Evolutionary analysis and functional identification of BPPS genes showed that BPP-related terpenoids may be distributed only in the Zingiberaceae of monocot plants. This research provides valuable genomic resources for breeding and improving Fructus Amomi with medicinal and edible value and sheds light on the evolution of terpenoid biosynthesis in Zingiberaceae.

Chromosome-level genome assembly and functional characterization of terpene synthases provide insights into the volatile terpenoid biosynthesis of Wurfbainia villosa.

Wurfbainia villosa is a well-known medicinal and edible plant that is widely cultivated in the Lingnan region of China. Its dried fruits (called Fructus Amomi) are broadly used in traditional Chinese medicine for curing gastrointestinal diseases and are rich in volatile terpenoids. Here, we report a high-quality chromosome-level genome assembly of W. villosa with a total size of approximately 2.80 Gb, 42 588 protein-coding genes, and a very high percentage of repetitive sequences (87.23%). Genome analysis showed that W. villosa likely experienced a recent whole-genome duplication event prior to the W. villosa-Zingiber officinale divergence (approximately 11 million years ago), and a recent burst of long terminal repeat insertions afterward. The W. villosa genome enabled the identification of 17 genes involved in the terpenoid skeleton biosynthesis pathway and 66 terpene synthase (TPS) genes. We found that tandem duplication events have an important contribution to the expansion of WvTPSs, which likely drove the production of volatile terpenoids. In addition, functional characterization of 18 WvTPSs, focusing on the TPS-a and TPS-b subfamilies, showed that most of these WvTPSs are multi-product TPS and are predominantly expressed in seeds. The present study provides insights into the genome evolution and the molecular basis of the volatile terpenoids diversity in W. villosa. The genome sequence also represents valuable resources for the functional gene research and molecular breeding of W. villosa.

Genome-Wide Identification of BAHD Superfamily and Functional Characterization of Bornyl Acetyltransferases Involved in the Bornyl Acetate Biosynthesis in Wurfbainia villosa.

Bornyl acetate (BA) is known as a natural aromatic monoterpene ester with a wide range of pharmacological and biological activities. Borneol acetyltransferase (BAT), catalyzing borneol and acetyl-CoA to synthesize BA, is alcohol acetyltransferase, which belongs to the BAHD super acyltransferase family, however, BAT, responsible for the biosynthesis of BA, has not yet been characterized. The seeds of Wurfbainia villosa (homotypic synonym: Amomum villosum) are rich in BA. Here we identified 64 members of the BAHD gene family from the genome of W. villosa using both PF02458 (transferase) and PF07247 (AATase) as Hidden Markov Model (HMM) to screen the BAHD genes. A total of sixty-four WvBAHDs are distributed on 14 chromosomes and nine unanchored contigs, clustering into six clades; three WvBAHDs with PF07247 have formed a separated and novel clade: clade VI. Twelve candidate genes belonging to clade I-a, I-b, and VI were selected to clone and characterize in vitro, among which eight genes have been identified to encode BATs acetylating at least one type of borneol to synthesize BA. All eight WvBATs can utilize (-)-borneol as substrates, but only five WvBATs can catalyze (+)-borneol, which is the endogenous borneol substrate in the seeds of W. villosa; WvBAT3 and WvBAT4 present the better catalytic efficiency on (+)-borneol than the others. The temporal and spatial expression patterns of WvBATs indicate that WvBAT3 and WvBAT4 are seed-specific expression genes, and their expression levels are correlated with the accumulation of BA, suggesting WvBAT3 and WvBAT4 might be the two key BATs for BA synthesis in the seeds of W. villosa. This is the first report on BAT responsible for the last biosynthetic step of BA, which will contribute to further studies on BA biosynthesis and metabolism engineering of BA in other plants or heterologous hosts.

Automatic Identification of Individual Primates with Deep Learning Techniques.

The difficulty of obtaining reliable individual identification of animals has limited researcher's ability to obtain quantitative data to address important ecological, behavioral, and conservation questions. Traditional marking methods placed animals at undue risk. Machine learning approaches for identifying species through analysis of animal images has been proved to be successful. But for many questions, there needs a tool to identify not only species but also individuals. Here, we introduce a system developed specifically for automated face detection and individual identification with deep learning methods using both videos and still-framed images that can be reliably used for multiple species. The system was trained and tested with a dataset containing 102,399 images of 1,040 individuals across 41 primate species whose individual identity was known and 6,562 images of 91 individuals across four carnivore species. For primates, the system correctly identified individuals 94.1% of the time and could process 31 facial images per second.

Seasonal Variation and Sexual Dimorphism of the Microbiota in Wild Blue Sheep (Pseudois nayaur).

Microbiota of the wild blue sheep (Pseudois nayaur) presents a seasonal variation due to different dietary selection and feeding strategies from different ecological niches chosen by different sex in summer. To address those issues, we analyzed the variation of gut microbiota based on the material from the feces, with 16S rRNA and meta-genome aimed to explore seasonal and gender differences. The results indicate that seasonal dietary changes and gender differentiation, as expected, cause the variation in sheep's gut microbiota structure. The variation of the former is more significant than the latter. Dominant Firmicutes exists a significantly higher abundance in summer than that in winter. Subordinate Bacteroides expresses no seasonal difference between the two seasons. Compared with the winter group, the summer group is featured by abundant enzymes digesting cellulose and generating short-chain fatty acids (SCFAs), such as beta-glucosidase (EC: 3.2.1.21) for cellulose digestion, and butyrate kinase (EC:2.7.2.7) in butyrate metabolism, implying that the changes of the composition in intestinal flora allow the sheep to adapt to the seasonalized dietary selection through alternated microbial functions to reach the goal of facilitating the efficiency of energy harvesting. The results also show that the blue sheep expresses a prominent sexual dimorphism in the components of gut microbiota, indicating that the two sexes have different adaptations to the dietary selection, and demands for physical and psychological purposes. Thus, this study provides an example of demonstrating the principles and regulations of natural selection and environmental adaptation.

Using unmanned aerial vehicles with thermal-image acquisition cameras for animal surveys: a case study on the Sichuan snub-nosed monkey in the Qinling Mountains.

Following significant developments in technology, alternative devices have been applied in fieldwork for animal and plant surveys. Thermal-image acquisition cameras installed on unmanned aerial vehicles (UAVs) have been used in animal surveys in the wilderness. This article demonstrates an example of how UAVs can be used in high mountainous regions, presenting a case study on the Sichuan snub-nosed monkey with a detection rate of 65.19% for positive individual identification. It also presents a model that can prospectively predict population size for a given animal species, which is based on combined initial work using UAVs and traditional surveys on the ground. A great potential advantage of UAVs is significantly shortening survey procedures, particularly for areas with high mountains and plateaus, such as the Himalayas, the Qinghai-Tibet Plateau, Hengduan Mountains, the Yunnan-Gui Plateau and Qinling Mountains in China, where carrying out a traditional survey is extremely difficult, so that species and population surveys, particularly for critically endangered animals, are largely absent. This lack of data has impacted the management of endangered animals as well as the formulation and amendment of conservation strategies.

Gut Microbiota of Wild and Captive Alpine Musk Deer (Moschus chrysogaster).

As for the wild animals, their diet components are always changed, so that we have to monitor such changes by analyzing the modification of intestinal microbial community. Such effort allows us to amend their conservation strategies and tactics accordingly so that they are able to appropriately adapt to the new environment and dietary selection. In this study we focus on the gut flora of two groups of an endangered species, Alpine musk deer (Moschus chrysogaster), wild group (WG) which is compared with that of the individuals of the same species but kept in the captivities (CG), a control group. Such a project is aimed to work out whether the composition of the gut microbes has significantly been changed due to captive feedings. To do so, we used 16S rRNA amplicon sequencing to characterize gut bacteria of the musk deer from the two groups. The results show that there is a significant difference in community structure of the bacteria: WG shows significant enrichment of Firmicutes and depletion of Bacteroidetes, while CG has a significant abundance of Proteobacteria and Euryarchaeota. Metagenomics was used to analyze the differences in functional enzymes between the two groups. The related results indicate that genes in WG are mostly related to the enzymes digesting cellulose and generating short-chain fatty acids (SCFAs) for signaling pathways, but CG shows enrichment in methanogenesis, including the CO2/H2 pathway and the methylotrophic pathway. Thus, this study indicates that the Firmicutes-rich gut microbiota in the WG enables individuals to maximize their energy intake from the cellulose, and has significant abundance of Euryarchaeota and methanogenesis pathways that allow them to reduce redundant energy consumption in methane metabolism, ensuring them to adapt to the wild environments.