Measuring calcium content in plants using NEXAFS spectroscopy.

Calcium is important for the growth and development of plants. It serves crucial functions in cell wall and cell membrane structure and serves as a secondary messenger in signaling pathways relevant to nutrient and immunity responses. Thus, measuring calcium levels in plants is important for studies of plant biology and for technology development in food, agriculture, energy, and forest industries. Often, calcium in plants has been measured through techniques such as atomic absorption spectrophotometry (AAS), inductively coupled plasma-mass spectrometry (ICP-MS), and electrophysiology. These techniques, however, require large sample sizes, chemical extraction of samples or have limited spatial resolution. Here, we used near-edge X-ray absorption fine structure (NEXAFS) spectroscopy at the calcium L- and K-edges to measure the calcium to carbon mass ratio with spatial resolution in plant samples without requiring chemical extraction or large sample sizes. We demonstrate that the integrated absorbance at the calcium L-edge and the edge jump in the fluorescence yield at the calcium K-edge can be used to quantify the calcium content as the calcium mass fraction, and validate this approach with onion epidermal peels and ICP-MS. We also used NEXAFS to estimate the calcium mass ratio in hypocotyls of a model plant, Arabidopsis thaliana, which has a cell wall composition that is similar to that of onion epidermal peels. These results show that NEXAFS spectroscopy performed at the calcium edge provides an approach to quantify calcium levels within plants, which is crucial for understanding plant physiology and advancing plant-based materials.

Enzymatic Synthesis of Xylan Microparticles with Tunable Morphologies.

Xylans are a diverse family of hemicellulosic polysaccharides found in abundance within the cell walls of nearly all flowering plants. Unfortunately, naturally occurring xylans are highly heterogeneous, limiting studies of their synthesis and structure-function relationships. Here, we demonstrate that xylan synthase 1 from the charophyte alga Klebsormidium flaccidum is a powerful biocatalytic tool for the bottom-up synthesis of pure ß-1,4 xylan polymers that self-assemble into microparticles in vitro. Using uridine diphosphate-xylose (UDP-xylose) and defined saccharide primers as substrates, we demonstrate that the shape, composition, and properties of the self-assembling xylan microparticles could be readily controlled via the fine structure of the xylan oligosaccharide primer used to initiate polymer elongation. Furthermore, we highlight two approaches for bottom-up and surface functionalization of xylan microparticles with chemical probes and explore the susceptibility of xylan microparticles to enzymatic hydrolysis. Together, these results provide a useful platform for structural and functional studies of xylans to investigate cell wall biosynthesis and polymer-polymer interactions and suggest possible routes to new biobased materials with favorable properties for biomedical and renewable applications.

Operation Blood Rain Phase 2: Evaluating the Effect of Airdrop on Fresh and Stored Whole Blood.

BACKGROUND: Transfusion of whole blood (WB) is a lifesaving treatment that prolongs life until definitive surgical intervention can be performed; however, collecting WB is a time-consuming and resource-intensive process. Furthermore, it may be difficult to collect sufficient WB at the point of injury to treat critically wounded patients or multiple hemorrhaging casualties. This study is a follow-up to the proof-of-concept study on the effect of airdrop on WB. In addition, this study confirms the statistical significance for the plausibility of using airdrop to deliver WB to combat medics treating casualties in the pre-hospital setting when Food and Drug Administration (FDA)-approved cold-stored blood products are not available. METHODS: Forty-eight units of WB were collected and loaded into a blood cooler that was dropped from a fixed-wing aircraft under a Standard Airdrop Training Bundle (SATB) parachute or 68-in pilot chute. Twenty-four of these units were dropped from a C-145 aircraft, and 24 were dropped from a C-130 aircraft. A control group of 15 units of WB was storedin a blood cooler that was not dropped. Baseline and post-intervention laboratory tests were measured in both airdroppedand control units, including complete blood count; prothrombin time/partial thromboplastin time (PT/PTT); pH, lactate,potassium, bilirubin, glucose, fibrinogen, and lactate dehydrogenase (LDH) levels; and peripheral blood smears. RESULTS: The blood cooler, cooling packs, and all 48 WB units did notsustain any major damage from the airdrop. There was noevidence of hemolysis. Except for the one slightly damagedbag that was not sampled, all airdropped blood met parameters for transfusion per the Joint Trauma System Whole BloodTransfusion Clinical Practice Guideline and the Associationfor the Advancement of Blood and Biotherapies (AABB) Circular of Information for the Use of Human Blood and BloodComponents. CONCLUSIONS: Airdrop of fresh or stored WB in ablood cooler with a chute is a viable way of delivering bloodproducts to combat medics treating hemorrhaging patientsin the pre-hospital setting. This study also demonstrated theportability of this technique for multiple aircraft. The techniques evaluated in this study have the potential for utilizationin other austere settings such as wilderness medicine or humanitarian disasters where an acute need for WB delivery by airdrop is the only option.

Polygalacturonase activity promotes aberrant cell separation in the quasimodo2 mutant of Arabidopsis thaliana.

In plants, cell adhesion relies on balancing the integrity of the pectin-rich middle lamella with wall loosening during tissue expansion. Mutation of QUASIMODO2 (QUA2), a pectin methyltransferase, causes defective hypocotyl elongation and cell adhesion in Arabidopsis thaliana hypocotyls. However, the molecular function of QUA2 in cell adhesion is obscured by complex genetic and environmental interactions. To dissect the role of QUA2 in cell adhesion, we investigated a qua2 loss-of-function mutant and a suppressor mutant with restored cell adhesion, qua2 esmeralda1, using a combination of imaging and biochemical techniques. We found that qua2 hypocotyls have reductions in middle lamellae integrity, pectin methyl-esterase (PME) activity, pectin content and molecular mass, and immunodetected Ca2+-crosslinking at cell corners, but increased methyl-esterification and polygalacturonase (PG) activity, with qua2 esmd1 having wild type-like or intermediate phenotypes. Our findings suggest that excessive pectin degradation prevents pectin accumulation and the formation of a sufficiently Ca2+-crosslinked network to maintain cell adhesion in qua2 mutants. We propose that PME and PG activities balance tissue-level expansion and cell separation. Together, these data provide insight into the cause of cell adhesion defects in qua2 mutants and highlight the importance of harmonizing pectin modification and degradation during plant growth and development.

Protocols for isolating and characterizing polysaccharides from plant cell walls: a case study using rhamnogalacturonan-II.

BACKGROUND: In plants, a large diversity of polysaccharides comprise the cell wall. Each major type of plant cell wall polysaccharide, including cellulose, hemicellulose, and pectin, has distinct structures and functions that contribute to wall mechanics and influence plant morphogenesis. In recent years, pectin valorization has attracted much attention due to its expanding roles in biomass deconstruction, food and material science, and environmental remediation. However, pectin utilization has been limited by our incomplete knowledge of its structure. Herein, we present a workflow of principles relevant for the characterization of polysaccharide primary structure using nature's most complex polysaccharide, rhamnogalacturonan-II (RG-II), as a model. RESULTS: We outline how to isolate RG-II from celery and duckweed cell walls and from red wine using chemical or enzymatic treatments coupled with size-exclusion chromatography. From there, we applied mass spectrometry (MS)-based techniques to determine the glycosyl residue and linkage compositions of the intact RG-II and derived oligosaccharides including special considerations for labile monosaccharides. In doing so, we demonstrated that in the duckweed Wolffiella repanda the arabinopyranosyl (Arap) residue of side chain B is substituted at O-2 with rhamnose. We used electrospray-MS techniques to identify non-glycosyl modifications including methyl-ethers, methyl-esters, and acetyl-esters on RG-II-derived oligosaccharides. We then showed the utility of proton nuclear magnetic resonance spectroscopy (1H-NMR) to investigate the structure of intact RG-II and to complement the RG-II dimerization studies performed using size-exclusion chromatography. CONCLUSIONS: The complexity of pectic polysaccharide structures has hampered efforts aimed at their valorization. In this work, we used RG-II as a model to demonstrate the steps necessary to isolate and characterize polysaccharides using chromatographic, MS, and NMR techniques. The principles can be applied to the characterization of other saccharide structures and will help inform researchers on how saccharide structure relates to functional properties in the future.

Mutations in the Pectin Methyltransferase QUASIMODO2 Influence Cellulose Biosynthesis and Wall Integrity in Arabidopsis.

Pectins are abundant in the cell walls of dicotyledonous plants, but how they interact with other wall polymers and influence wall integrity and cell growth has remained mysterious. Here, we verified that QUASIMODO2 (QUA2) is a pectin methyltransferase and determined that QUA2 is required for normal pectin biosynthesis. To gain further insight into how pectin affects wall assembly and integrity maintenance, we investigated cellulose biosynthesis, cellulose organization, cortical microtubules, and wall integrity signaling in two mutant alleles of Arabidopsis (Arabidopsis thaliana) QUA2, qua2 and tsd2 In both mutants, crystalline cellulose content is reduced, cellulose synthase particles move more slowly, and cellulose organization is aberrant. NMR analysis shows higher mobility of cellulose and matrix polysaccharides in the mutants. Microtubules in mutant hypocotyls have aberrant organization and depolymerize more readily upon treatment with oryzalin or external force. The expression of genes related to wall integrity, wall biosynthesis, and microtubule stability is dysregulated in both mutants. These data provide insights into how homogalacturonan is methylesterified upon its synthesis, the mechanisms by which pectin functionally interacts with cellulose, and how these interactions are translated into intracellular regulation to maintain the structural integrity of the cell wall during plant growth and development.

Preferred crystallographic orientation of cellulose in plant primary cell walls.

Cellulose, the most abundant biopolymer on earth, is a versatile, energy rich material found in the cell walls of plants, bacteria, algae, and tunicates. It is well established that cellulose is crystalline, although the orientational order of cellulose crystallites normal to the plane of the cell wall has not been characterized. A preferred orientational alignment of cellulose crystals could be an important determinant of the mechanical properties of the cell wall and of cellulose-cellulose and cellulose-matrix interactions. Here, the crystalline structures of cellulose in primary cell walls of onion (Allium cepa), the model eudicot Arabidopsis (Arabidopsis thaliana), and moss (Physcomitrella patens) were examined through grazing incidence wide angle X-ray scattering (GIWAXS). We find that GIWAXS can decouple diffraction from cellulose and epicuticular wax crystals in cell walls. Pole figures constructed from a combination of GIWAXS and X-ray rocking scans reveal that cellulose crystals have a preferred crystallographic orientation with the (200) and (110)/([Formula: see text]) planes preferentially stacked parallel to the cell wall. This orientational ordering of cellulose crystals, termed texturing in materials science, represents a previously unreported measure of cellulose organization and contradicts the predominant hypothesis of twisting of microfibrils in plant primary cell walls.

Heterologous expression of plant glycosyltransferases for biochemistry and structural biology.

Much of the carbon captured by photosynthesis is converted into the polysaccharides that constitute plant cell walls. These complex macrostructures are composed of cellulose, hemicellulose, and pectins, together with small amounts of structural proteins, minerals, and in many cases lignin. Wall components assemble and interact with one another to produce dynamic structures with many capabilities, including providing mechanical support to plant structures and determining plant cell shape and size. Despite their abundance, major gaps in our knowledge of the synthesis of the building blocks of these polymers remain, largely due to ineffective methods for expression and purification of active synthetic enzymes for in vitro biochemical analyses. The hemicellulosic polysaccharide, xyloglucan, comprises up to 25% of the dry weight of primary cell walls in plants. Most of the knowledge about the glycosyltransferases (GTs) involved in the xyloglucan biosynthetic pathway has been derived from the identification and carbohydrate analysis of knockout mutants, lending little information on how the catalytic biosynthesis of xyloglucan occurs in planta. In this chapter we describe methods for the heterologous expression of plant GTs using the HEK293 expression platform. As a demonstration of the utility of this platform, nine xyloglucan-relevant GTs from three different CAZy families were evaluated, and methods for expression, purification, and construct optimization are described for biochemical and structural characterization.

Imaging the delivery and behavior of cellulose synthases in Arabidopsis thaliana using confocal microscopy.

Confocal microscopy has been a key tool for characterizing the behavior of cellulose synthase (CESA) proteins as they extrude cellulose into the apoplast to help construct plant cell walls. While other microscopy techniques like electron microscopy can achieve higher resolution images of CESAs, confocal microscopy is still the most accessible way to image these proteins in living plants as they are trafficked to and from the cell surface and move through the plasma membrane. Here, we describe a method for imaging fluorescently tagged CESA proteins in seedlings of Arabidopsis thaliana using spinning disk confocal microscopy, with a focus on quantifying the speed, density, and delivery rate of CESA particles. Many of these techniques can be adapted and applied to imaging other membrane-localized proteins and other plant species. In addition to imaging techniques, we describe several options for image analysis that can be optimized for different datasets.

Biochemical and Genetic Analysis Identify CSLD3 as a beta-1,4-Glucan Synthase That Functions during Plant Cell Wall Synthesis.

In plants, changes in cell size and shape during development fundamentally depend on the ability to synthesize and modify cell wall polysaccharides. The main classes of cell wall polysaccharides produced by terrestrial plants are cellulose, hemicelluloses, and pectins. Members of the cellulose synthase (CESA) and cellulose synthase-like (CSL) families encode glycosyltransferases that synthesize the ß-1,4-linked glycan backbones of cellulose and most hemicellulosic polysaccharides that comprise plant cell walls. Cellulose microfibrils are the major load-bearing component in plant cell walls and are assembled from individual ß-1,4-glucan polymers synthesized by CESA proteins that are organized into multimeric complexes called CESA complexes, in the plant plasma membrane. During distinct modes of polarized cell wall deposition, such as in the tip growth that occurs during the formation of root hairs and pollen tubes or de novo formation of cell plates during plant cytokinesis, newly synthesized cell wall polysaccharides are deposited in a restricted region of the cell. These processes require the activity of members of the CESA-like D subfamily. However, while these CSLD polysaccharide synthases are essential, the nature of the polysaccharides they synthesize has remained elusive. Here, we use a combination of genetic rescue experiments with CSLD-CESA chimeric proteins, in vitro biochemical reconstitution, and supporting computational modeling and simulation, to demonstrate that Arabidopsis (Arabidopsis thaliana) CSLD3 is a UDP-glucose-dependent ß-1,4-glucan synthase that forms protein complexes displaying similar ultrastructural features to those formed by CESA6.

SHOU4 Proteins Regulate Trafficking of Cellulose Synthase Complexes to the Plasma Membrane.

Cell walls play critical roles in plants, regulating tissue mechanics, defining the extent and orientation of cell expansion, and providing a physical barrier against pathogen attack [1]. Cellulose microfibrils, which are synthesized by plasma membrane-localized cellulose synthase (CESA) complexes, are the primary load-bearing elements of plant cell walls [2]. Cell walls are dynamic structures that are regulated in part by cell wall integrity (CWI)-monitoring systems that feed back to modulate wall properties and the synthesis of new wall components [3]. Several receptor-like kinases have been implicated as sensors of CWI [3-5], including the FEI1/FEI2 receptor-like kinases [4]. Here, we characterize two genes encoding novel plant-specific plasma membrane proteins (SHOU4 and SHOU4L) that were identified in a suppressor screen of the cellulose-deficient fei1 fei2 mutant. shou4 shou4l double mutants display phenotypes consistent with elevated levels of cellulose, and elevated levels of non-crystalline cellulose are present in this mutant. Disruption of SHOU4 and SHOU4L increases the abundance of CESA proteins at the plasma membrane as a result of enhanced exocytosis. The SHOU4/4L N-terminal cytosolic domains directly interact with CESAs. Our results suggest that the SHOU4 proteins regulate cellulose synthesis in plants by influencing the trafficking of CESA complexes to the cell surface.

Cytosolic invertases contribute to cellulose biosynthesis and influence carbon partitioning in seedlings of Arabidopsis thaliana.

In plants, UDP-glucose is the direct precursor for cellulose biosynthesis, and can be converted into other NDP-sugars required for the biosynthesis of wall matrix polysaccharides. UDP-glucose is generated from sucrose by two distinct metabolic pathways. The first pathway is the direct conversion of sucrose to UDP-glucose and fructose by sucrose synthase. The second pathway involves sucrose hydrolysis by cytosolic invertase (CINV), conversion of glucose to glucose-6-phosphate and glucose-1-phosphate, and UDP-glucose generation by UDP-glucose pyrophosphorylase (UGP). Previously, Barratt et al. (Proc. Natl Acad. Sci. USA, 106, 2009 and 13124) have found that an Arabidopsis double mutant lacking CINV1 and CINV2 displayed drastically reduced growth. Whether this reduced growth is due to deficient cell wall production caused by limited UDP-glucose supply, pleiotropic effects, or both, remained unresolved. Here, we present results indicating that the CINV/UGP pathway contributes to anisotropic growth and cellulose biosynthesis in Arabidopsis. Biochemical and imaging data demonstrate that cinv1 cinv2 seedlings are deficient in UDP-glucose production, exhibit abnormal cellulose biosynthesis and microtubule properties, and have altered cellulose organization without substantial changes to matrix polysaccharide composition, suggesting that the CINV/UGP pathway is a key metabolic route to UDP-glucose synthesis in Arabidopsis. Furthermore, differential responses of cinv1 cinv2 seedlings to exogenous sugar supplementation support a function of CINVs in influencing carbon partitioning in Arabidopsis. From these data and those of previous studies, we conclude that CINVs serve central roles in cellulose biosynthesis and carbon allocation in Arabidopsis.

Longevity in vivo of primary cell wall cellulose synthases.

KEY MESSAGE: Our work focuses on understanding the lifetime and thus stability of the three main cellulose synthase (CESA) proteins involved in primary cell wall synthesis of Arabidopsis. It had long been thought that a major means of CESA regulation was via their rapid degradation. However, our studies here have uncovered that AtCESA proteins are not rapidly degraded. Rather, they persist for an extended time in the plant cell. Plant cellulose is synthesized by membrane-embedded cellulose synthase complexes (CSCs). The CSC is composed of cellulose synthases (CESAs), of which three distinct isozymes form the primary cell wall CSC and another set of three isozymes form the secondary cell wall CSC. We determined the stability over time of primary cell wall (PCW) CESAs in Arabidopsis thaliana seedlings, using immunoblotting after inhibiting protein synthesis with cycloheximide treatment. Our work reveals very slow turnover for the Arabidopsis PCW CESAs in vivo. Additionally, we show that the stability of all three CESAs within the PCW CSC is altered by mutations in individual CESAs, elevated temperature, and light conditions. Together, these results suggest that CESA proteins are very stable in vivo, but that their lifetimes can be modulated by intrinsic and environmental cues.

Release, Recycle, Rebuild: Cell-Wall Remodeling, Autodegradation, and Sugar Salvage for New Wall Biosynthesis during Plant Development.

Plant cell walls contain elaborate polysaccharide networks and regulate plant growth, development, mechanics, cell-cell communication and adhesion, and defense. Despite conferring rigidity to support plant structures, the cell wall is a dynamic extracellular matrix that is modified, reorganized, and degraded to tightly control its properties during growth and development. Far from being a terminal carbon sink, many wall polymers can be degraded and recycled by plant cells, either via direct re-incorporation by transglycosylation or via internalization and metabolic salvage of wall-derived sugars to produce new precursors for wall synthesis. However, the physiological and metabolic contributions of wall recycling to plant growth and development are largely undefined. In this review, we discuss long-standing and recent evidence supporting the occurrence of cell-wall recycling in plants, make predictions regarding the developmental processes to which wall recycling might contribute, and identify outstanding questions and emerging experimental tools that might be used to address these questions and enhance our understanding of this poorly characterized aspect of wall dynamics and metabolism.

Acetyl Bromide Soluble Lignin (ABSL) Assay for Total Lignin Quantification from Plant Biomass.

Lignin is the second most abundant biopolymer on Earth, providing plants with mechanical support in secondary cell walls and defense against abiotic and biotic stresses. However, lignin also acts as a barrier to biomass saccharification for biofuel generation (Carroll and Somerville, 2009; Zhao and Dixon, 2011; Wang et al., 2013 ). For these reasons, studying the properties of lignin is of great interest to researchers in agriculture and bioenergy fields. This protocol describes the acetyl bromide method of total lignin extraction and quantification, which is favored among other methods for its high recovery, consistency, and insensitivity to different tissue types ( Johnson et al., 1961 ; Chang et al., 2008 ; Moreira- Vilar et al., 2014 ; Kapp et al., 2015 ). In brief, acetyl bromide digestion causes the formation of acetyl derivatives on free hydroxyl groups and bromide substitution of α-carbon hydroxyl groups on the lignin backbone to cause a complete solubilization of lignin, which can be quantified using known extinction coefficients and absorbance at 280 nm (Moreira- Vilar et al., 2014 ).

Activation tagging of Arabidopsis POLYGALACTURONASE INVOLVED IN EXPANSION2 promotes hypocotyl elongation, leaf expansion, stem lignification, mechanical stiffening, and lodging.

Pectin is the most abundant component of primary cell walls in eudicot plants. The modification and degradation of pectin affects multiple processes during plant development, including cell expansion, organ initiation, and cell separation. However, the extent to which pectin degradation by polygalacturonases affects stem development and secondary wall formation remains unclear. Using an activation tag screen, we identified a transgenic Arabidopsis thaliana line with longer etiolated hypocotyls, which overexpresses a gene encoding a polygalacturonase. We designated this gene as POLYGALACTURONASE INVOLVED IN EXPANSION2 (PGX2), and the corresponding activation tagged line as PGX2AT . PGX2 is widely expressed in young seedlings and in roots, stems, leaves, flowers, and siliques of adult plants. PGX2-GFP localizes to the cell wall, and PGX2AT plants show higher total polygalacturonase activity and smaller pectin molecular masses than wild-type controls, supporting a function for this protein in apoplastic pectin degradation. A heterologously expressed, truncated version of PGX2 also displays polygalacturonase activity in vitro. Like previously identified PGX1AT plants, PGX2AT plants have longer hypocotyls and larger rosette leaves, but they also uniquely display early flowering, earlier stem lignification, and lodging stems with enhanced mechanical stiffness that is possibly due to decreased stem thickness. Together, these results indicate that PGX2 both functions in cell expansion and influences secondary wall formation, providing a possible link between these two developmental processes.

Imaging with the fluorogenic dye Basic Fuchsin reveals subcellular patterning and ecotype variation of lignification in Brachypodium distachyon.

Lignin is a complex polyphenolic heteropolymer that is abundant in the secondary cell walls of plants and functions in growth and defence. It is also a major barrier to the deconstruction of plant biomass for bioenergy production, but the spatiotemporal details of how lignin is deposited in actively lignifying tissues and the precise relationships between wall lignification in different cell types and developmental events, such as flowering, are incompletely understood. Here, the lignin-detecting fluorogenic dye, Basic Fuchsin, was adapted to enable comparative fluorescence-based imaging of lignin in the basal internodes of three Brachypodium distachyon ecotypes that display divergent flowering times. It was found that the extent and intensity of Basic Fuchsin fluorescence increase over time in the Bd21-3 ecotype, that Basic Fuchsin staining is more widespread and intense in 4-week-old Bd21-3 and Adi-10 basal internodes than in Bd1-1 internodes, and that Basic Fuchsin staining reveals subcellular patterns of lignin in vascular and interfascicular fibre cell walls. Basic Fuchsin fluorescence did not correlate with lignin quantification by acetyl bromide analysis, indicating that whole-plant and subcellular lignin analyses provide distinct information about the extent and patterns of lignification in B. distachyon. Finally, it was found that flowering time correlated with a transient increase in total lignin, but did not correlate strongly with the patterning of stem lignification, suggesting that additional developmental pathways might regulate secondary wall formation in grasses. This study provides a new comparative tool for imaging lignin in plants and helps inform our views of how lignification proceeds in grasses.

Population genetics of freeze tolerance among natural populations of Populus balsamifera across the growing season.

Protection against freeze damage during the growing season influences the northern range limits of plants. Freeze tolerance and freeze avoidance are the two major freeze resistance strategies. Winter survival strategies have been extensively studied in perennials, but few have addressed them and their genetic basis during the growing season. We examined intraspecific phenotypic variation in freeze resistance of Populus balsamifera across latitude and the growing season. To investigate the molecular basis of this variation, we surveyed nucleotide diversity and examined patterns of gene expression in the poplar C-repeat binding factor (CBF) gene family. Foliar freeze tolerance exhibited latitudinal and seasonal variation indicative of natural genotypic variation. CBF6 showed signatures of recent selective sweep. Of the 46 SNPs surveyed across the six CBF homologs, only CBF2_619 exhibited latitudinal differences consistent with increased freeze tolerance in the north. All six CBF genes were cold inducible, but showed varying patterns of expression across the growing season. Some Poplar CBF homologs exhibited patterns consistent with historical selection and clinal variation in freeze tolerance documented here. However, the CBF genes accounted for only a small amount of the variation, indicating that other genes in this and other molecular pathways likely play significant roles in nature.

An ATP binding cassette transporter is required for cuticular wax deposition and desiccation tolerance in the moss Physcomitrella patens.

The plant cuticle is thought to be a critical evolutionary adaptation that allowed the first plants to colonize land, because of its key roles in regulating plant water status and providing protection from biotic and abiotic stresses. Much has been learned about cuticle composition and structure through genetic and biochemical studies of angiosperms, as well as underlying genetic pathways, but little is known about the cuticles of early diverging plant lineages. Here, we demonstrate that the moss Physcomitrella patens, an extant relative of the earliest terrestrial plants, has a cuticle that is analogous in both structure and chemical composition to those of angiosperms. To test whether the underlying cuticle biosynthetic pathways were also shared among distant plant lineages, we generated a genetic knockout of the moss ATP binding cassette subfamily G (ABCG) transporter Pp-ABCG7, a putative ortholog of Arabidopsis thaliana ABCG transporters involved in cuticle precursor trafficking. We show that this mutant is severely deficient in cuticular wax accumulation and has a reduced tolerance of desiccation stress compared with the wild type. This work provides evidence that the cuticle was an adaptive feature present in the first terrestrial plants and that the genes involved in their formation have been functionally conserved for over 450 million years.

Rapid identification of dengue virus by reverse transcription-polymerase chain reaction using field-deployable instrumentation.

Dengue virus universal and dengue serotype 1 to 4, fluorogenic probe hydrolysis (TaqMan), reverse transcription-polymerase chain reaction assays were developed for screening and serotype identification of infected mosquito vectors and human sera using a field-deployable, fluorometric thermocycler. Dengue universal and dengue 1 to 4 serotype assay in vitro sensitivity and specificity results were 100% concordant when tested with total nucleic acid extracts of multiple strains of dengue serotype 1 to 4, yellow fever, Japanese encephalitis, West Nile, and St. Louis encephalitis viruses. The in vitro sensitivity and specificity results for all five assays were concordant when tested with a blind panel of 27 dengue virus-infected mosquitoes, 21 non-dengue (yellow fever, West Nile, or St. Louis encephalitis) flavivirus-infected mosquitoes, and 11 uninfected mosquitoes and with clinical specimens consisting of a human serum panel of eight dengue viremic and 31 non-dengue-infected febrile patient serum samples. No cross-reaction occurred with vector species or human genomic DNA. Sample processing and polymerase chain reaction required <2 hours.