The selection landscape and genetic legacy of ancient Eurasians.

The Holocene (beginning around 12,000 years ago) encompassed some of the most significant changes in human evolution, with far-reaching consequences for the dietary, physical and mental health of present-day populations. Using a dataset of more than 1,600 imputed ancient genomes1, we modelled the selection landscape during the transition from hunting and gathering, to farming and pastoralism across West Eurasia. We identify key selection signals related to metabolism, including that selection at the FADS cluster began earlier than previously reported and that selection near the LCT locus predates the emergence of the lactase persistence allele by thousands of years. We also find strong selection in the HLA region, possibly due to increased exposure to pathogens during the Bronze Age. Using ancient individuals to infer local ancestry tracts in over 400,000 samples from the UK Biobank, we identify widespread differences in the distribution of Mesolithic, Neolithic and Bronze Age ancestries across Eurasia. By calculating ancestry-specific polygenic risk scores, we show that height differences between Northern and Southern Europe are associated with differential Steppe ancestry, rather than selection, and that risk alleles for mood-related phenotypes are enriched for Neolithic farmer ancestry, whereas risk alleles for diabetes and Alzheimer's disease are enriched for Western hunter-gatherer ancestry. Our results indicate that ancient selection and migration were large contributors to the distribution of phenotypic diversity in present-day Europeans.

Biosynthesis of selenium-containing small molecules in diverse microorganisms.

Selenium is an essential micronutrient in diverse organisms. Two routes are known for its insertion into proteins and nucleic acids, via selenocysteine and 2-selenouridine, respectively1. However, despite its importance, pathways for specific incorporation of selenium into small molecules have remained elusive. Here we use a genome-mining strategy in various microorganisms to uncover a widespread three-gene cluster that encodes a dedicated pathway for producing selenoneine, the selenium analogue of the multifunctional molecule ergothioneine2,3. We elucidate the reactions of all three proteins and uncover two novel selenium-carbon bond-forming enzymes and the biosynthetic pathway for production of a selenosugar, which is an unexpected intermediate en route to the final product. Our findings expand the scope of biological selenium utilization, suggest that the selenometabolome is more diverse than previously thought, and set the stage for the discovery of other selenium-containing natural products.

Genome-Wide Identification, Characterization and Expression Analysis of the CIPK Gene Family in Potato (Solanum tuberosum L.) and the Role of StCIPK10 in Response to Drought and Osmotic Stress.

The potato (Solanum tuberosum L.), one of the most important food crops worldwide, is sensitive to environmental stresses. Sensor-responder complexes comprising calcineurin B-like (CBL) proteins and CBL-interacting protein kinases (CIPKs) not only modulate plant growth and development but also mediate numerous stress responses. Here, using a Hidden Markov Model and BLAST searches, 27 CIPK genes were identified in potato and divided into five groups by phylogenetic analysis and into two clades (intron-poor and intron-rich) by gene structure analysis. Quantitative reverse-transcription PCR (qRT-PCR) assays revealed that StCIPK genes play important roles in plant growth, development and abiotic stress tolerance. Up-regulated expression of StCIPK10 was significantly induced by drought, PEG6000 and ABA. StCIPK10 enhances both the ability of potato to scavenge reactive oxygen species and the content of corresponding osmoregulation substances, thereby strengthening tolerance to drought and osmotic stress. StCIPK10 is located at the intersection between the abscisic acid and abiotic stress signaling pathways, which control both root growth and stomatal closure in potato. In addition, StCIPK10 interacts with StCBL1, StCBL4, StCBL6, StCBL7, StCBL8, StCBL11 and StCBL12, and is specifically recruited to the plasma membrane by StCBL11.

The Laccase Gene Family Mediate Multi-Perspective Trade-Offs during Tea Plant (Camellia sinensis) Development and Defense Processes.

Laccase (LAC) plays important roles in different plant development and defense processes. In this study, we identified laccase genes (CsLACs) in Camellia sinensis cv 'Longjing43' cultivars, which were classified into six subclades. The expression patterns of CsLACs displayed significant spatiotemporal variations across different tissues and developmental stages. Most members in subclades II, IV and subclade I exhibited contrasting expression patterns during leaf development, consistent with a trade-off model for preferential expression in the early and late developmental stages. The extensive transcriptional changes of CsLACs under different phytohormone and herbivore treatment were observed and compared, with the expression of most genes in subclades I, II and III being downregulated but genes in subclades IV, V and VI being upregulated, suggesting a growth and defense trade-off model between these subclades. Taken together, our research reveal that CsLACs mediate multi-perspective trade-offs during tea plant development and defense processes and are involved in herbivore resistance in tea plants. More in-depth research of CsLACs upstream regulation and downstream targets mediating herbivore defense should be conducted in the future.

In-silico genome wide analysis of Mitogen activated protein kinase kinase kinase gene family in C. sinensis.

Mitogen activated protein kinase kinase kinase (MAPKKK) form the upstream component of MAPK cascade. It is well characterized in several plants such as Arabidopsis and rice however the knowledge about MAPKKKs in tea plant is largely unknown. In the present study, MAPKKK genes of tea were obtained through a genome wide search using Arabidopsis thaliana as the reference genome. Among 59 candidate MAPKKK genes in tea, 17 genes were MEKK-like, 31 genes were Raf-like and 11 genes were ZIK- like. Additionally, phylogenetic relationships were established along with structural analysis, which includes gene structure, its location as well as conserved motifs, cis-acting regulatory elements and functional domain signatures that were systematically examined. Also, on the basis of one orthologous gene found between tea and Arabidopsis, functional interaction was carried out in C. sinensis based on an Arabidopsis association model. The expressional profiles indicated major involvement of MAPKKK genes from tea in response to various abiotic stress factors. Taken together, this study provides the targets for additional inclusive identification, functional study, and provides comprehensive knowledge for a better understanding of the MAPKKK cascade regulatory network in C. sinensis.

Expansion within the CYP71D subfamily drives the heterocyclization of tanshinones synthesis in Salvia miltiorrhiza.

Tanshinones are the bioactive nor-diterpenoid constituents of the Chinese medicinal herb Danshen (Salvia miltiorrhiza). These groups of chemicals have the characteristic furan D-ring, which differentiates them from the phenolic abietane-type diterpenoids frequently found in the Lamiaceae family. However, how the 14,16-epoxy is formed has not been elucidated. Here, we report an improved genome assembly of Danshen using a highly homozygous genotype. We identify a cytochrome P450 (CYP71D) tandem gene array through gene expansion analysis. We show that CYP71D373 and CYP71D375 catalyze hydroxylation at carbon-16 (C16) and 14,16-ether (hetero)cyclization to form the D-ring, whereas CYP71D411 catalyzes upstream hydroxylation at C20. In addition, we discover a large biosynthetic gene cluster associated with tanshinone production. Collinearity analysis indicates a more specific origin of tanshinones in Salvia genus. It illustrates the evolutionary origin of abietane-type diterpenoids and those with a furan D-ring in Lamiaceae.

Genome-wide identification and expression analysis of the GSK gene family in Solanum tuberosum L. under abiotic stress and phytohormone treatments and functional characterization of StSK21 involvement in salt stress.

Plant Glycogen Synthase Kinase 3 (GSK3)/SHAGGY-like kinase (GSK) proteins play important roles in modulating growth, development, and stress responses in several plant species. However, little is known about the members of the potato GSK (StGSK) family. Here, nine StGSK genes were identified and phylogenetically grouped into four clades. Gene duplication analysis revealed that segmental duplication contributed to the expansion of the StGSK family. Gene structure and motif pattern analyses indicated that similar exon/intron and motif organizations were found in StGSKs from the same clade. Conserved motif and kinase activity analyses indicated that the StGSKs encode active protein kinases, and they were shown to be distributed throughout whole cells. Cis-acting regulatory element analysis revealed the presence of many growth-, hormone-, and stress-responsive elements within the promoter regions of the StGSKs, which is consistent with their expression in different organs, and their altered expression in response to hormone and stress treatments. Association network analysis indicated that various proteins, including two confirmed BES1 family transcription factors, potentially interact with StGSKs. Overexpression of StSK21 provides enhanced sensitivity to salt stress in Arabidopsis thaliana plants. Overall, these results reveal that StGSK proteins are active protein kinases with purported functions in regulating growth, development, and stress responses.

Pantoea ananatis carotenoid production confers toxoflavin tolerance and is regulated by Hfq-controlled quorum sensing.

Carotenoids are widely used in functional foods, cosmetics, and health supplements, and their importance and scope of use are continuously expanding. Here, we characterized carotenoid biosynthetic genes of the plant-pathogenic bacterium Pantoea ananatis, which carries a carotenoid biosynthetic gene cluster (including crtE, X, Y, I, B, and Z) on a plasmid. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that the crtEXYIB gene cluster is transcribed as a single transcript and crtZ is independently transcribed in the opposite direction. Using splicing by overlap extension with polymerase chain reaction (SOE by PCR) based on asymmetric amplification, we reassembled crtE-B, crtE-B-I, and crtE-B-I-Y. High-performance liquid chromatography confirmed that Escherichia coli expressing the reassembled crtE-B, crtE-B-I, and crtE-B-I-Y operons produced phytoene, lycopene, and ß-carotene, respectively. We found that the carotenoids conferred tolerance to UV radiation and toxoflavin. Pantoea ananatis shares rice environments with the toxoflavin producer Burkholderia glumae and is considered to be the first reported example of producing and using carotenoids to withstand toxoflavin. We confirmed that carotenoid production by P. ananatis depends on RpoS, which is positively regulated by Hfq/ArcZ and negatively regulated by ClpP, similar to an important regulatory network of E. coli (HfqArcZ →RpoS Í° ClpXP). We also demonstrated that Hfq-controlled quorum signaling de-represses EanR to activate RpoS, thereby initiating carotenoid production. Survival genes such as those responsible for the production of carotenoids of the plant-pathogenic P. ananatis must be expressed promptly to overcome stressful environments and compete with other microorganisms. This mechanism is likely maintained by a brake with excellent performance, such as EanR.

Evolution of a plant gene cluster in Solanaceae and emergence of metabolic diversity.

Plants produce phylogenetically and spatially restricted, as well as structurally diverse specialized metabolites via multistep metabolic pathways. Hallmarks of specialized metabolic evolution include enzymatic promiscuity and recruitment of primary metabolic enzymes and examples of genomic clustering of pathway genes. Solanaceae glandular trichomes produce defensive acylsugars, with sidechains that vary in length across the family. We describe a tomato gene cluster on chromosome 7 involved in medium chain acylsugar accumulation due to trichome specific acyl-CoA synthetase and enoyl-CoA hydratase genes. This cluster co-localizes with a tomato steroidal alkaloid gene cluster and is syntenic to a chromosome 12 region containing another acylsugar pathway gene. We reconstructed the evolutionary events leading to this gene cluster and found that its phylogenetic distribution correlates with medium chain acylsugar accumulation across the Solanaceae. This work reveals insights into the dynamics behind gene cluster evolution and cell-type specific metabolite diversity.

Plants produce a vast variety of different molecules known as secondary or specialized metabolites to attract pollinating insects, such as bees, or protect themselves against herbivores and pests. The secondary metabolites are made from simple building blocks that are readily available in plants, including amino acids, fatty acids and sugars. Different species of plant, and even different parts of the same plant, produce their own sets of secondary metabolites. For example, the hairs on the surface of tomatoes and other members of the nightshade family of plants make metabolites known as acylsugars. These chemicals deter herbivores and pests from damaging the plants. To make acylsugars, the plants attach long chains known as fatty acyl groups to molecules of sugar, such as sucrose. Some members of the nightshade family produce acylsugars with longer chains than others. In particular, acylsugars with long chains are only found in tomatoes and other closely-related species. It remained unclear how the nightshade family evolved to produce acylsugars with chains of different lengths. To address this question, Fan et al. used genetic and biochemical approaches to study tomato plants and other members of the nightshade family. The experiments identified two genes known as AACS and AECH in tomatoes that produce acylsugars with long chains. These two genes originated from the genes of older enzymes that metabolize fatty acids ­ the building blocks of fats ­ in plant cells. Unlike the older genes, AACS and AECH were only active at the tips of the hairs on the plant's surface. Fan et al. then investigated the evolutionary relationship between 11 members of the nightshade family and two other plant species. This revealed that AACS and AECH emerged in the nightshade family around the same time that longer chains of acylsugars started appearing. These findings provide insights into how plants evolved to be able to produce a variety of secondary metabolites that may protect them from a broader range of pests. The gene cluster identified in this work could be used to engineer other species of crop plants to start producing acylsugars as natural pesticides.

Evaluation of mtr cluster expression in Shewanella RCRI7 during uranium removal.

In recent years, bioremediation is considered as an efficient method to remove the pollutants from the industrial wastewater. In this study, quantitative gene expressions (Real-time RT-PCR) of mtr gene cluster (mtrA, mtrB, mtrC, mtrD, mtrE, mtrF and omcA) in five different uranium concentrations (0.1, 0.25, 0.5, 1 and 2 mM) were performed with ICP and microscopic live cell counting analysis under anaerobic condition, by Shewanella RCRI7 as a native bacterium. The results indicated that the amount of uranium removal and live-cell counting were decreased in the higher uranium concentrations (1 and 2 mM), due to the uranium toxicity, suggesting 0.5 mM as the optimum uranium concentration for Shewanella RCRI7 resistance. The expression of mtrCED and omcA genes presented increasing trend in the lower uranium concentrations (0.1, 0.25 and 0.5 mM) and a decreasing trend in 1 and 2 mM, while mtrABF, presented an inverse pattern, proving the alternative role of mtrF for mtrC and omcA, as the substantial multiheme cytochromes in Extracellular Electron Transfer (EET) pathway. These data are a proof of these gene vital roles in the EET pathway, proposing them for genetic engineering toward EET optimization, as the certain pathway in heavy metal bioremediation process.

The bHLH gene family and its response to saline stress in Jilin ginseng, Panax ginseng C.A. Meyer.

Basic helix-loop-helix (bHLH) gene family is a gene family of transcription factors that plays essential roles in plant growth and development, secondary metabolism and response to biotic and abiotic stresses. Therefore, a comprehensive knowledge of the bHLH gene family is paramount to understand the molecular mechanisms underlying these processes and develop advanced technologies to manipulate the processes efficiently. Ginseng, Panax ginseng C.A. Meyer, is a well-known medicinal herb; however, little is known  about the bHLH genes (PgbHLH) in the species. Here, we identified 137 PgbHLH genes from Jilin ginseng cultivar, Damaya, widely cultivated in Jilin, China, of which 50 are newly identified by pan-genome analysis. These 137 PgbHLH genes were phylogenetically classified into 26 subfamilies, suggesting their sequence diversification. They are alternatively spliced into 366 transcripts in a 4-year-old plant and involved in 11 functional subcategories of the gene ontology, indicating their functional differentiation in ginseng. The expressions of the PgbHLH genes dramatically vary spatio-temporally and across 42 genotypes, but they are still somehow functionally correlated. Moreover, the PgbHLH gene family, at least some of its genes, is shown to have roles in plant response to the abiotic stress of saline. These results provide a new insight into the evolution and functional differentiation of the bHLH gene family in plants, new bHLH genes to the PgbHLH gene family, and saline stress-responsive genes for genetic improvement in ginseng and other plant species.

The Microbiome: A Reservoir to Discover New Antimicrobials Agents.

Nature offered mankind the first golden era of discovery of novel antimicrobials based on the ability of eukaryotes or micro-organisms to produce such compounds. The microbial world proved to be a huge reservoir of such antimicrobial compounds which play important functional roles in every environment. However, most of those organisms are still uncultivable in a classical way, and therefore, the use of extended culture or DNA based methods (metagenomics) to discover novel compounds promises usefulness. In the past decades, the advances in next-generation sequencing and bioinformatics revealed the enormous diversity of the microbial worlds and the functional repertoire available for studies. Thus, data-mining becomes of particular interest in the context of the increased need for new antibiotics due to antimicrobial resistance and the rush in antimicrobial discovery. In this review, an overview of principles will be presented to discover new natural compounds from the microbiome. We describe culture-based and culture-independent (metagenomic) approaches that have been developed to identify new antimicrobials and the input of those methods in the field as well as their limitations.

Genome-wide identification and expression analysis of the superoxide dismutase (SOD) gene family in Salvia miltiorrhiza.

Adverse environmental conditions, such as salinity, cold, drought, heavy metals, and pathogens affect the yield and quality of Salvia miltiorrhiza, a well-known medicinal plant used for the treatment of cardiovascular and cerebrovascular diseases. Superoxide dismutase (SOD), a key enzyme of antioxidant system in plants, plays a vital role in protecting plants against various biotic and abiotic stresses via scavenging the reactive oxygen species produced by organisms. However, little is known about the SOD gene family in S. miltiorrhiza. In this study, eight SOD genes, including three Cu/Zn-SODs, two Fe-SODs and three Mn-SODs, were identified in the S. miltiorrhiza genome. Their gene structures, promoters, protein features, phylogenetic relationships, and expression profiles were comprehensively investigated. Gene structure analysis implied that most SmSODs have different introns/exons distrbution patterns. Many cis-elements related to different stress responses or plant hormones were found in the promoter of each SmSOD. Expression profile analysis indicated that SmSODs exhibited diverse responses to cold, salt, drought, heavy metal, and plant hormones. Additionally, 31 types of TFs regulating SmSODs were predicted and analyzed. These findings provided valuable information for further researches on the functions and applications of SmSODs in S. miltiorrhiza growth and adaptation to stress.

Effects of Coffee Intake on Dyslipidemia Risk According to Genetic Variants in the ADORA Gene Family among Korean Adults.

Current evidence on the effects of coffee intake on cardiovascular diseases is not consistent, in part contributed by the genetic variability of the study subjects. While adenosine receptors (ADORAs) are involved in caffeine signaling, it remains unknown how genetic variations at the ADORA loci correlate the coffee intake with cardiovascular diseases. The present study examined the associations of coffee intake with dyslipidemia risk depending on genetic variants in the ADORA gene family. The study involved a population-based cohort of 4898 Korean subjects. Consumption of more than or equal to a cup of coffee per day was associated with lower dyslipidemia risk in females carrying the ADORA2B minor allele rs2779212 (OR: 0.645, 95% CI: 0.506-0.823), but not in those with the major allele. At the ADORA2A locus, male subjects with the minor allele of rs5760423 showed instead an increased risk of dyslipidemia when consuming more than or equal to a cup of coffee per day (OR: 1.352, 95% CI: 1.014-1.802). The effect of coffee intake on dyslipidemia risk differs depending on genetic variants at the ADORA loci in a sex-specific manner. Our study suggests that a dietary guideline for coffee intake in the prevention and management of dyslipidemia ought to consider ADORA-related biomarkers carefully.

Genome-wide and expression pattern analysis of JAZ family involved in stress responses and postharvest processing treatments in Camellia sinensis.

The JASMONATE-ZIM DOMAIN (JAZ) family genes are key repressors in the jasmonic acid signal transduction pathway. Recently, the JAZ gene family has been systematically characterized in many plants. However, this gene family has not been explored in the tea plant. In this study, 13 CsJAZ genes were identified in the tea plant genome. Phylogenetic analysis showed that the JAZ proteins from tea and other plants clustered into 11 sub-groups. The CsJAZ gene transcriptional regulatory network predictive and expression pattern analyses suggest that these genes play vital roles in abiotic stress responses, phytohormone crosstalk and growth and development of the tea plant. In addition, the CsJAZ gene expression profiles were associated with tea postharvest processing. Our work provides a comprehensive understanding of the CsJAZ family and will help elucidate their contributions to tea quality during tea postharvest processing.

Analysis of Terpene Synthase Family Genes in Camellia sinensis with an Emphasis on Abiotic Stress Conditions.

For a better understanding terpenoid volatile production in Camellia sinensis, global terpenoid synthase gene (TPS) transcription analysis was conducted based on transcriptomic data combined with terpenoid metabolic profiling under different abiotic stress conditions. Totally 80 TPS-like genes were identified. Twenty-three CsTPS genes possessed a complete coding sequence and most likely were functional. The remaining 57 in the currently available database lack essential gene structure or full-length transcripts. Distinct tempo-spatial expression patterns of CsTPS genes were found in tea plants. 17 genes were substantially expressed in all the tested organs with a few exceptions. The other 17 were predominantly expressed in leaves whereas additional eight were primarily expressed in flowers. Under the treatments of cold acclimation, salt and polyethylene glycol, CsTPS67, -69 and -71 were all suppressed and the inhibited expression of many others were found in multiple stress treatments. However, methyl jasmonate resulted in the enhanced expression of the majority of CsTPS genes. These transcription data were largely validated using qPCR. Moreover, volatile terpenoid profiling with leaves, flowers and stress-treated plants revealed a general association between the abundances of mono- and sesqui-terpenoids and some CsTPS genes. These results provide vital information for future studies on CsTPS regulation of terpenoid biosynthesis.

Genome-Wide Study of the GATL Gene Family in Gossypium hirsutum L. Reveals that GhGATL Genes Act on Pectin Synthesis to Regulate Plant Growth and Fiber Elongation.

Pectin is a major polysaccharide component that promotes plant growth and fiber elongation in cotton. In previous studies, the galacturonosyltransferase-like (GATL) gene family has been shown to be involved in pectin synthesis. However, few studies have been performed on cotton GATL genes. Here, a total of 33, 17, and 16 GATL genes were respectively identified in Gossypium hirsutum, Gossypium raimondii, and Gossypium arboreum. In multiple plant species, phylogenetic analysis divided GATL genes into five groups named GATL-a to GATL-e, and the number of groups was found to gradually change over evolution. Whole genome duplication (WGD) and segmental duplication played a significant role in the expansion of the GATL gene family in G. hirsutum. Selection pressure analyses revealed that GATL-a and GATL-b groups underwent a great positive selection pressure during evolution. Moreover, the expression patterns revealed that most of highly expressed GhGATL genes belong to GATL-a and GATL-b groups, which have more segmental duplications and larger positive selection value, suggesting that these genes may play an important role in the evolution of cotton plants. We overexpressed GhGATL2, GhGATL9, GhGATL12, and GhGATL15 in Arabidopsis and silenced the GhGATL15 gene in cotton through a virus induced gene silencing assay (VIGS). The transgenic and VIGS lines showed significant differences in stem diameter, epidermal hair length, stamen length, seed size, and fiber length than the control plant. In addition, the pectin content test proved that the pectin was significantly increased in the transgenic lines and reduced in VIGS plants, demonstrating that GhGATL genes have similar functions and act on the pectin synthesis to regulate plant growth and fiber elongation. In summary, we performed a comprehensive analysis of GhGATL genes in G. hirsutum including evolution, structure and function, in order to better understand GhGATL genes in cotton for further studies.

Genome-wide analysis and expression profiles of the StR2R3-MYB transcription factor superfamily in potato (Solanum tuberosum L.).

MYB transcription factors comprise one of the largest families in plant kingdom, which play a variety of functions in plant developmental processes and defence responses, the R2R3-MYB members are the predominant form found in higher plants. In the present study, a total of 111 StR2R3-MYB transcription factors were identified and further phylogenetically classified into 31 subfamilies, as supported by highly conserved gene structures and motifs. Collinearity analysis showed that the segmental duplication events played a crucial role in the expansion of StR2R3-MYB gene family. Synteny analysis indicated that 37 and 13 StR2R3-MYB genes were orthologous to Arabidopsis and wheat (Triticum aestivum), respectively, and these gene pairs have evolved under strong purifying selection. RNA-seq data from different tissues and abiotic stresses revealed tissue-preferential and abiotic stress-responsive StR2R3-MYB genes. We further analyzed StR2R3-MYB genes might be involved in anthocyanin biosynthesis and drought stress by using RNA-seq data of pigmented tetraploid potato cultivars and drought-sensitive and -tolerant tetraploid potato cultivars under drought stress, respectively. Moreover, EAR motifs were found in 21 StR2R3-MYB proteins and 446 pairs of proteins were predicted to interact with 21 EAR motif-containing StR2R3-MYB proteins by constructing the interaction network with medium confidence (0.4). Additionally, Gene Ontology (GO) analysis of the 21 EAR motif-containing StR2R3-MYB proteins was performed to further investigate their functions. This work will facilitate future biologically functional studies of potato StR2R3-MYB transcription factors and enrich the knowledge of MYB superfamily genes in plant species.

Mutually Regulated AP2/ERF Gene Clusters Modulate Biosynthesis of Specialized Metabolites in Plants.

APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) gene clusters regulate the biosynthesis of diverse specialized metabolites, including steroidal glycoalkaloids in tomato (Solanum lycopersicum) and potato (Solanum tuberosum), nicotine in tobacco (Nicotiana tabacum), and pharmaceutically valuable terpenoid indole alkaloids in Madagascar periwinkle (Catharanthus roseus). However, the regulatory relationships between individual AP2/ERF genes within the cluster remain unexplored. We uncovered intracluster regulation of the C. roseus AP2/ERF regulatory circuit, which consists of ORCA3, ORCA4, and ORCA5 ORCA3 and ORCA5 activate ORCA4 by directly binding to a GC-rich motif in the ORCA4 promoter. ORCA5 regulates its own expression through a positive autoregulatory loop and indirectly activates ORCA3 In determining the functional conservation of AP2/ERF clusters in other plant species, we found that GC-rich motifs are present in the promoters of analogous AP2/ERF clusters in tobacco, tomato, and potato. Intracluster regulation is evident within the tobacco NICOTINE2 (NIC2) ERF cluster. Moreover, overexpression of ORCA5 in tobacco and of NIC2 ERF189 in C. roseus hairy roots activates nicotine and terpenoid indole alkaloid pathway genes, respectively, suggesting that the AP2/ERFs are functionally equivalent and are likely to be interchangeable. Elucidation of the intracluster and mutual regulation of transcription factor gene clusters advances our understanding of the underlying molecular mechanism governing regulatory gene clusters in plants.

Structure, gene cluster of the O antigen and biological activity of the lipopolysaccharide from the rhizospheric bacterium Ochrobactrum cytisi IPA7.2.

Lipopolysaccharide (LPS) of Ochrobactrum cytisi IPA7.2, a bacterium isolated from the roots of Solanum tuberosum L., was extracted from dry bacterial cells and chemically characterized. The O-specific polysaccharide was obtained by mild acid hydrolysis of the LPS and studied by sugar analysis and 1H and 13C NMR spectroscopy, including 1H,1H COSY, 1H,1H TOCSY, 1H,1H ROESY, 1H,13C HSQC, and 1H,13C HMBC experiments. The polysaccharide was linear and consisted of trisaccharide repeating units of the following structure: A putative O-antigen gene cluster of O. cytisi IPA7.2 was identified and found to be consistent with the O-specific polysaccharide structure. The LPS of Ochrobactrum cytisi IPA7.2 promoted the growth of potato microplants in vitro.