Mononuclear phagocyte morphological response to chemoattractants is dependent on geranylgeranyl pyrophosphate.

Statins are used to treat hypercholesterolemia and function by inhibiting the production of the rate-limiting metabolite mevalonate. As such, statin treatment not only inhibits de novo synthesis of cholesterol but also isoprenoids that are involved in prenylation, the posttranslational lipid modification of proteins. The immunomodulatory effects of statins are broad and often conflicting. Previous work demonstrated that statins increased survival and inhibited myeloid cell trafficking in a murine model of sepsis, but the exact mechanisms underlying this phenomenon were unclear. Herein, we investigated the role of prenylation in chemoattractant responses. We found that simvastatin treatment abolished chemoattractant responses induced by stimulation by C5a and FMLP. The inhibitory effect of simvastatin treatment was unaffected by the addition of either farnesyl pyrophosphate (FPP) or squalene but was reversed by restoring geranylgeranyl pyrophosphate (GGPP). Treatment with prenyltransferase inhibitors showed that the chemoattractant response to both chemoattractants was dependent on geranylgeranylation. Proteomic analysis of C15AlkOPP-prenylated proteins identified several geranylgeranylated proteins involved in chemoattractant responses, including RHOA, RAC1, CDC42, and GNG2. Chemoattractant responses in THP-1 human macrophages were also geranylgeranylation dependent. These studies provide data that help clarify paradoxical findings on the immunomodulatory effects of statins. Furthermore, they establish the role of geranylgeranylation in mediating the morphological response to chemoattractant C5a.NEW & NOTEWORTHY The immunomodulatory effect of prenylation is ill-defined. We investigated the role of prenylation on the chemoattractant response to C5a. Simvastatin treatment inhibits the cytoskeletal remodeling associated with a chemotactic response. We showed that the chemoattractant response to C5a was dependent on geranylgeranylation, and proteomic analysis identified several geranylgeranylated proteins that are involved in C5a receptor signaling and cytoskeletal remodeling. Furthermore, they establish the role of geranylgeranylation in mediating the response to chemoattractant C5a.

A dolabralexin-deficient mutant provides insight into specialized diterpenoid metabolism in maize.

Two major groups of specialized metabolites in maize (Zea mays), termed kauralexins and dolabralexins, serve as known or predicted diterpenoid defenses against pathogens, herbivores, and other environmental stressors. To consider the physiological roles of the recently discovered dolabralexin pathway, we examined dolabralexin structural diversity, tissue-specificity, and stress-elicited production in a defined biosynthetic pathway mutant. Metabolomics analyses support a larger number of dolabralexin pathway products than previously known. We identified dolabradienol as a previously undetected pathway metabolite and characterized its enzymatic production. Transcript and metabolite profiling showed that dolabralexin biosynthesis and accumulation predominantly occur in primary roots and show quantitative variation across genetically diverse inbred lines. Generation and analysis of CRISPR-Cas9-derived loss-of-function Kaurene Synthase-Like 4 (Zmksl4) mutants demonstrated dolabralexin production deficiency, thus supporting ZmKSL4 as the diterpene synthase responsible for the conversion of geranylgeranyl pyrophosphate precursors into dolabradiene and downstream pathway products. Zmksl4 mutants further display altered root-to-shoot ratios and root architecture in response to water deficit. Collectively, these results demonstrate dolabralexin biosynthesis via ZmKSL4 as a committed pathway node biochemically separating kauralexin and dolabralexin metabolism, and suggest an interactive role of maize dolabralexins in plant vigor during abiotic stress.

Discovery, Biosynthesis and Stress-Related Accumulation of Dolabradiene-Derived Defenses in Maize.

Terpenoids are a major component of maize (Zea mays) chemical defenses that mediate responses to herbivores, pathogens, and other environmental challenges. Here, we describe the biosynthesis and elicited production of a class of maize diterpenoids, named dolabralexins. Dolabralexin biosynthesis involves the sequential activity of two diterpene synthases, ENT-COPALYL DIPHOSPHATE SYNTHASE (ZmAN2) and KAURENE SYNTHASE-LIKE4 (ZmKSL4). Together, ZmAN2 and ZmKSL4 form the diterpene hydrocarbon dolabradiene. In addition, we biochemically characterized a cytochrome P450 monooxygenase, ZmCYP71Z16, which catalyzes the oxygenation of dolabradiene to yield the epoxides 15,16-epoxydolabrene (epoxydolabrene) and 3ß-hydroxy-15,16-epoxydolabrene (epoxydolabranol). The absence of dolabradiene and epoxydolabranol in Zman2 mutants under elicited conditions confirmed the in vivo biosynthetic requirement of ZmAN2. Combined mass spectrometry and NMR experiments demonstrated that much of the epoxydolabranol is further converted into 3ß,15,16-trihydroxydolabrene (trihydroxydolabrene). Metabolite profiling of field-grown maize root tissues indicated that dolabralexin biosynthesis is widespread across common maize cultivars, with trihydroxydolabrene as the predominant diterpenoid. Oxidative stress induced dolabralexin accumulation and transcript expression of ZmAN2 and ZmKSL4 in root tissues, and metabolite and transcript accumulation were up-regulated in response to elicitation with the fungal pathogens Fusarium verticillioides and Fusarium graminearum Consistently, epoxydolabranol significantly inhibited the growth of both pathogens in vitro at 10 µg mL-1, while trihydroxydolabrene-mediated inhibition was specific to Fverticillioides These findings suggest that dolabralexins have defense-related roles in maize stress interactions and expand the known chemical space of diterpenoid defenses as genetic targets for understanding and ultimately improving maize resilience.

Chloroplasts in anther endothecium of Zea mays (Poaceae).

PREMISE OF THE STUDY: Although anthers of Zea mays, Oryza sativa, and Arabidopsis thaliana have been studied intensively using genetic and biochemical analyses in the past 20 years, few updates to anther anatomical and ultrastructural descriptions have been reported. For example, no transmission electron microscopy (TEM) images of the premeiotic maize anther have been published. Here we report the presence of chloroplasts in maize anthers. METHODS: TEM imaging, electron acceptor photosynthesis assay, in planta photon detection, microarray analysis, and light and fluorescence microscopy were used to investigate the presence of chloroplasts in the maize anther. KEY RESULTS: Most cells of the maize subepidermal endothecium have starch-containing chloroplasts that do not conduct measurable photosynthesis in vitro. CONCLUSIONS: The maize anther contains chloroplasts in most subepidermal, endothecial cells. Although maize anthers receive sufficient light to photosynthesize in vivo and the maize anther transcribes >96% of photosynthesis-associated genes found in the maize leaf, no photosynthetic light reaction activity was detected in vitro. The endothecial cell layer should no longer be defined as a complete circle viewed transversely in anther lobes, because chloroplasts are observed only in cells directly beneath the epidermis and not those adjacent to the connective tissue. We propose that chloroplasts be a defining characteristic of differentiated endothecial cells and that nonsubepidermal endothecial cells that lack chloroplasts be defined as a separate cell type, the interendothecium.

Deep Learning in Image-Based Plant Phenotyping.

A major bottleneck in the crop improvement pipeline is our ability to phenotype crops quickly and efficiently. Image-based, high-throughput phenotyping has a number of advantages because it is nondestructive and reduces human labor, but a new challenge arises in extracting meaningful information from large quantities of image data. Deep learning, a type of artificial intelligence, is an approach used to analyze image data and make predictions on unseen images that ultimately reduces the need for human input in computation. Here, we review the basics of deep learning, assessments of deep learning success, examples of applications of deep learning in plant phenomics, best practices, and open challenges.

Optimized Methods for Applying and Assessing Heat, Drought, and Nutrient Stress of Maize Seedlings in Controlled Environment Experiments.

Maize (Zea mays), also known as corn, is an important crop that plays a crucial role in global agriculture. The economic uses of maize are numerous, including for food, feed, fiber, and fuel. It has had a significant historical importance in research as well, with important discoveries made in maize regarding plant domestication, transposons, heterosis, genomics, and epigenetics. Unfortunately, environmental stresses cause substantial yield loss to maize crops each year. Yield losses are predicted to increase in future climate scenarios, posing a threat to food security and other sectors of the global economy. Developing efficient methods to study maize abiotic stress responses is a crucial step toward a more resilient and productive agricultural system. This review describes the importance of and methods for studying the effects of heat, drought, and nutrient deficiency on early developmental stages of maize grown in controlled environments. Studying the early effects of environmental stressors in controlled environments allows researchers to work with a variety of environmental conditions with low environmental variance, which can inform future field-based research. We highlight the current knowledge of physiological responses of maize to heat, drought, and nutrient stress; remaining knowledge gaps and challenges; and information on how standardized protocols can address these issues.

Maize Abiotic Stress Treatments in Controlled Environments.

Maize (Zea mays) is one of the world's most important crops, providing food for humans and livestock and serving as a bioenergy source. Climate change and the resulting abiotic stressors in the field reduce crop yields, threatening food security and the global economy. Water deficit (i.e., drought), heat, and insufficient nutrients (e.g., nitrogen and phosphorus) are major environmental stressors that affect maize yields, and impact growth and development at all stages of the plant life cycle. Understanding the biological processes underlying these responses in maize has the potential to increase yields in the face of abiotic stress. Optimizing individual or combined abiotic stress treatments in controlled environments reduces potential noise in data collection that can be present under less controlled growth conditions. Here, we describe methods and conditions for controlled abiotic stress treatments and associated controls during early vegetative growth of maize, conducted in greenhouses or growth chambers. This includes the environmental conditions, equipment, soil preparation, and intensity and duration of heat, drought, nitrogen deficiency, and phosphorous deficiency. Controlled experiments at early growth stages are informative for future in-field studies that require greater labor and inputs, saving researchers time and growing space, and thus research funds, before testing plants across later stages of development. We suggest that stress treatments be severe enough to result in a measurable phenotype, but not so severe that all plants die prior to sample collection. This protocol is designed to set important standards for replicable research in maize.

Bioactive diterpenoids impact the composition of the root-associated microbiome in maize (Zea mays).

Plants deploy both primary and species-specific, specialized metabolites to communicate with other organisms and adapt to environmental challenges, including interactions with soil-dwelling microbial communities. However, the role of specialized metabolites in modulating plant-microbiome interactions often remains elusive. In this study, we report that maize (Zea mays) diterpenoid metabolites with known antifungal bioactivities also influence rhizosphere bacterial communities. Metabolite profiling showed that dolabralexins, antibiotic diterpenoids that are highly abundant in roots of some maize varieties, can be exuded from the roots. Comparative 16S rRNA gene sequencing determined the bacterial community composition of the maize mutant Zman2 (anther ear 2), which is deficient in dolabralexins and closely related bioactive kauralexin diterpenoids. The Zman2 rhizosphere microbiome differed significantly from the wild-type sibling with the most significant changes observed for Alphaproteobacteria of the order Sphingomonadales. Metabolomics analyses support that these differences are attributed to the diterpenoid deficiency of the Zman2 mutant, rather than other large-scale metabolome alterations. Together, these findings support physiological functions of maize diterpenoids beyond known chemical defenses, including the assembly of the rhizosphere microbiome.

Specialized diterpenoid metabolism in monocot crops: Biosynthesis and chemical diversity.

Among the myriad specialized metabolites that plants employ to mediate interactions with their environment, diterpenoids form a chemically diverse group with vital biological functions. A few broadly abundant diterpenoids serve as core pathway intermediates in plant general metabolism. The majority of plant diterpenoids, however, function in specialized metabolism as often species-specific chemical defenses against herbivores and microbial diseases, in below-ground allelopathic interactions, as well as abiotic stress responses. Dynamic networks of anti-microbial diterpenoids were first demonstrated in rice (Oryza sativa) over four decades ago, and more recently, unique diterpenoid blends with demonstrated antibiotic bioactivities were also discovered in maize (Zea mays). Enabled by advances in -omics and biochemical approaches, species-specific diterpenoid-diversifying enzymes have been identified in these and other Poaceous species, including wheat (Triticum aestivum) and switchgrass (Panicum virgatum), and are discussed in this article with an emphasis on the critical diterpene synthase and cytochrome P450 monooxygenase families and their products. The continued investigation of the biosynthesis, diversity, and function of terpenoid-mediated crop defenses provides foundational knowledge to enable the development of strategies for improving crop resistance traits in the face of impeding pest, pathogen, and climate pressures impacting global agricultural production.

Genetic elucidation of interconnected antibiotic pathways mediating maize innate immunity.

Specialized metabolites constitute key layers of immunity that underlie disease resistance in crops; however, challenges in resolving pathways limit our understanding of the functions and applications of these metabolites. In maize (Zea mays), the inducible accumulation of acidic terpenoids is increasingly considered to be a defence mechanism that contributes to disease resistance. Here, to understand maize antibiotic biosynthesis, we integrated association mapping, pan-genome multi-omic correlations, enzyme structure-function studies and targeted mutagenesis. We define ten genes in three zealexin (Zx) gene clusters that encode four sesquiterpene synthases and six cytochrome P450 proteins that collectively drive the production of diverse antibiotic cocktails. Quadruple mutants in which the ability to produce zealexins (ZXs) is blocked exhibit a broad-spectrum loss of disease resistance. Genetic redundancies ensuring pathway resiliency to single null mutations are combined with enzyme substrate promiscuity, creating a biosynthetic hourglass pathway that uses diverse substrates and in vivo combinatorial chemistry to yield complex antibiotic blends. The elucidated genetic basis of biochemical phenotypes that underlie disease resistance demonstrates a predominant maize defence pathway and informs innovative strategies for transferring chemical immunity between crops.

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products.

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological functions in developmental processes, interorganismal interactions, and environmental adaptation. Due to these various bioactivities, many diterpenoids are also of economic importance as pharmaceuticals, food additives, biofuels, and other bioproducts. Advanced genomics and biochemical approaches have enabled a rapid increase in the knowledge of diterpenoid-metabolic genes, enzymes, and pathways. However, the structural complexity of diterpenoids and the narrow taxonomic distribution of individual compounds in often only a single species remain constraining factors for their efficient production. Availability of a broader range of metabolic enzymes now provide resources for producing diterpenoids in sufficient titers and purity to facilitate a deeper investigation of this important metabolite group. Drawing on established tools for microbial and plant-based enzyme co-expression, we present an easily operated and customizable protocol for the enzymatic production of diterpenoids in either Escherichia coli or Nicotiana benthamiana, and the purification of desired products via silica chromatography and semi-preparative HPLC. Using the group of maize (Zea mays) dolabralexin diterpenoids as an example, we highlight how modular combinations of diterpene synthase (diTPS) and cytochrome P450 monooxygenase (P450) enzymes can be used to generate different diterpenoid scaffolds. Purified compounds can be used in various downstream applications, such as metabolite structural analyses, enzyme structure-function studies, and in vitro and in planta bioactivity experiments.

Multiple genes recruited from hormone pathways partition maize diterpenoid defences.

Duplication and divergence of primary pathway genes underlie the evolution of plant specialized metabolism; however, mechanisms partitioning parallel hormone and defence pathways are often speculative. For example, the primary pathway intermediate ent-kaurene is essential for gibberellin biosynthesis and is also a proposed precursor for maize antibiotics. By integrating transcriptional coregulation patterns, genome-wide association studies, combinatorial enzyme assays, proteomics and targeted mutant analyses, we show that maize kauralexin biosynthesis proceeds via the positional isomer ent-isokaurene formed by a diterpene synthase pair recruited from gibberellin metabolism. The oxygenation and subsequent desaturation of ent-isokaurene by three promiscuous cytochrome P450s and a new steroid 5α reductase indirectly yields predominant ent-kaurene-associated antibiotics required for Fusarium stalk rot resistance. The divergence and differential expression of pathway branches derived from multiple duplicated hormone-metabolic genes minimizes dysregulation of primary metabolism via the circuitous biosynthesis of ent-kaurene-related antibiotics without the production of growth hormone precursors during defence.

Functional Characterization of Two Class II Diterpene Synthases Indicates Additional Specialized Diterpenoid Pathways in Maize (Zea mays).

As a major staple food, maize (Zea mays) is critical to food security. Shifting environmental pressures increasingly hamper crop defense capacities, causing expanded harvest loss. Specialized labdane-type diterpenoids are key components of maize chemical defense and ecological adaptation. Labdane diterpenoid biosynthesis most commonly requires the pairwise activity of class II and class I diterpene synthases (diTPSs) that convert the central precursor geranylgeranyl diphosphate into distinct diterpenoid scaffolds. Two maize class II diTPSs, ANTHER EAR 1 and 2 (ZmAN1/2), have been previously identified as catalytically redundant ent-copalyl diphosphate (CPP) synthases. ZmAN1 is essential for gibberellin phytohormone biosynthesis, whereas ZmAN2 is stress-inducible and governs the formation of defensive kauralexin and dolabralexin diterpenoids. Here, we report the biochemical characterization of the two remaining class II diTPSs present in the maize genome, COPALYL DIPHOSPHATE SYNTHASE 3 (ZmCPS3) and COPALYL DIPHOSPHATE SYNTHASE 4 (ZmCPS4). Functional analysis via microbial co-expression assays identified ZmCPS3 as a (+)-CPP synthase, with functionally conserved orthologs occurring in wheat (Triticum aestivum) and numerous dicot species. ZmCPS4 formed the unusual prenyl diphosphate, 8,13-CPP (labda-8,13-dien-15-yl diphosphate), as verified by mass spectrometry and nuclear magnetic resonance. As a minor product, ZmCPS4 also produced labda-13-en-8-ol diphosphate (LPP). Root gene expression profiles did not indicate an inducible role of ZmCPS3 in maize stress responses. By contrast, ZmCPS4 showed a pattern of inducible gene expression in roots exposed to oxidative stress, supporting a possible role in abiotic stress responses. Identification of the catalytic activities of ZmCPS3 and ZmCPS4 clarifies the first committed reactions controlling the diversity of defensive diterpenoids in maize, and suggests the existence of additional yet undiscovered diterpenoid pathways.

Reliability of a structured assessment for nonclinicians to detect delirium among new admissions to postacute care.

OBJECTIVE: To evaluate the interrater reliability of a structured delirium assessment method for nonclinician interviewers in elderly patients newly admitted for postacute care. DESIGN: Prospective assessment using dyads of nonclinician raters. SETTING: Postacute (Medicare) units at 6 skilled nursing facilities. PARTICIPANTS: Forty elderly patients newly admitted for postacute care from medical or surgical units at acute care hospitals. MEASUREMENTS: Subjects underwent dual delirium assessments within 5 days of admission. The standardized delirium assessment included the Mini-Mental Status Exam and Digit Span to assess overall cognitive function, the Delirium Symptom Interview to elicit specific delirium symptoms, the Memorial Delirium Assessment Scale to measure the severity of delirium, and the Confusion Assessment Method (CAM) to make the diagnosis of delirium. A coding protocol that linked observations to specific coding was used to improve reliability. RESULTS: The structured delirium assessment process produced very high interobserver agreement for all instruments. Kappa for agreement on delirium diagnosis was 0.95. CONCLUSIONS: Nonclinician interviewers using a structured delirium assessment achieved reliability that rivaled or exceeded that of trained clinical assessors in other studies. Nonclinicians may offer an effective alternative for the assessment of delirium among postacute patients in skilled nursing facilities.

Igniting the fire within: a primer on political advocacy for pharmacy professionals.

Due to the expanding role of pharmacy in health care, it is imperative that pharmacy professionals work together to advocate for the profession. An English-language only literature search was conducted of the PubMed and Medline databases using the key words advocacy, grassroots, political action committee, lobbying, politics, political action, legislation, letter writing, pharmacy, pharmacist, Capitol Hill. Up-to-date information regarding pharmacy-specific advocacy was limited and difficult to locate. Information from the literature search was supplemented with interviews of professionals actively engaged in advocacy, personal experience, and Web sites of national pharmacy organizations. This primer ignites the fire for political advocacy within pharmacy professionals by reinforcing the significant impact that advocacy has on the profession and by providing information on how to become involved. The primer provides a comprehensive "pocket guide" of resources combined into an easy-to-use reference for pharmacy professionals and outlines a structured approach on how to become a pharmacy advocate, ranging from a minimal level of involvement to master political activist, and to promote interest among pharmacy professionals to become more engaged with advocacy. Even a small act of advocacy or volunteerism can transform a spark into a raging fire.