Plant traits and vegetation data from climate warming experiments along an 1100 m elevation gradient in Gongga Mountains, China.

Functional trait data enhance climate change research by linking climate change, biodiversity response, and ecosystem functioning, and by enabling comparison between systems sharing few taxa. Across four sites along a 3000-4130 m a.s.l. gradient spanning 5.3 °C in growing season temperature in Mt. Gongga, Sichuan, China, we collected plant functional trait and vegetation data from control plots, open top chambers (OTCs), and reciprocally transplanted vegetation turfs. Over five years, we recorded vascular plant composition in 140 experimental treatment and control plots. We collected trait data associated with plant resource use, growth, and life history strategies (leaf area, leaf thickness, specific leaf area, leaf dry matter content, leaf C, N and P content and C and N isotopes) from local populations and from experimental treatments. The database consists of 6,671 plant records and 36,743 trait measurements (increasing the trait data coverage of the regional flora by 500%) covering 11 traits and 193 plant taxa (ca. 50% of which have no previous published trait data) across 37 families.

Leveraging CyVerse Resources for De Novo Comparative Transcriptomics of Underserved (Non-model) Organisms.

This workflow allows novice researchers to leverage advanced computational resources such as cloud computing to carry out pairwise comparative transcriptomics. It also serves as a primer for biologists to develop data scientist computational skills, e.g. executing bash commands, visualization and management of large data sets. All command line code and further explanations of each command or step can be found on the wiki (https://wiki.cyverse.org/wiki/x/dgGtAQ). The Discovery Environment and Atmosphere platforms are connected together through the CyVerse Data Store. As such, once the initial raw sequencing data has been uploaded there is no more need to transfer large data files over an Internet connection, minimizing the amount of time needed to conduct analyses. This protocol is designed to analyze only two experimental treatments or conditions. Differential gene expression analysis is conducted through pairwise comparisons, and will not be suitable to test multiple factors. This workflow is also designed to be manual rather than automated. Each step must be executed and investigated by the user, yielding a better understanding of data and analytical outputs, and therefore better results for the user. Once complete, this protocol will yield de novo assembled transcriptome(s) for underserved (non-model) organisms without the need to map to previously assembled reference genomes (which are usually not available in underserved organism). These de novo transcriptomes are further used in pairwise differential gene expression analysis to investigate genes differing between two experimental conditions. Differentially expressed genes are then functionally annotated to understand the genetic response organisms have to experimental conditions. In total, the data derived from this protocol is used to test hypotheses about biological responses of underserved organisms.

FractBias: a graphical tool for assessing fractionation bias following polyploidy.

Summary: Following polyploidy events, genomes undergo massive reduction in gene content through a process known as fractionation. Importantly, the fractionation process is not always random, and a bias as to which homeologous chromosome retains or loses more genes can be observed in some species. The process of characterizing whole genome fractionation requires identifying syntenic regions across genomes followed by post-processing of those syntenic datasets to identify and plot gene retention patterns. We have developed a tool, FractBias, to calculate and visualize gene retention and fractionation patterns across whole genomes. Through integration with SynMap and its parent platform CoGe, assembled genomes are pre-loaded and available for analysis, as well as letting researchers integrate their own data with security options to keep them private or make them publicly available. Availability and Implementation: FractBias is freely available as a web application at https://genomevolution.org/CoGe/SynMap.pl . The software is open source (MIT license) and executable with Python 2.7 or iPython notebook, and available on GitHub ( https://goo.gl/PaAtqy ). Documentation for FractBias is available on CoGepedia ( https://goo.gl/ou9dt6 ). Contact: ericlyons@email.arizona.edu. Supplementary information: Supplementary data are available at Bioinformatics online.

Resources and Recommendations for Using Transcriptomics to Address Grand Challenges in Comparative Biology.

High-throughput RNA sequencing (RNA-seq) technology has become an important tool for studying physiological responses of organisms to changes in their environment. De novo assembly of RNA-seq data has allowed researchers to create a comprehensive catalog of genes expressed in a tissue and to quantify their expression without a complete genome sequence. The contributions from the "Tapping the Power of Crustacean Transcriptomics to Address Grand Challenges in Comparative Biology" symposium in this issue show the successes and limitations of using RNA-seq in the study of crustaceans. In conjunction with the symposium, the Animal Genome to Phenome Research Coordination Network collated comments from participants at the meeting regarding the challenges encountered when using transcriptomics in their research. Input came from novices and experts ranging from graduate students to principal investigators. Many were unaware of the bioinformatics analysis resources currently available on the CyVerse platform. Our analysis of community responses led to three recommendations for advancing the field: (1) integration of genomic and RNA-seq sequence assemblies for crustacean gene annotation and comparative expression; (2) development of methodologies for the functional analysis of genes; and (3) information and training exchange among laboratories for transmission of best practices. The field lacks the methods for manipulating tissue-specific gene expression. The decapod crustacean research community should consider the cherry shrimp, Neocaridina denticulata, as a decapod model for the application of transgenic tools for functional genomics. This would require a multi-investigator effort.

Effects of Produced Water on Soil Characteristics, Plant Biomass, and Secondary Metabolites.

The Powder River Basin in Wyoming and Montana contains the United States' largest coal reserve. The area produces large amounts of natural gas through extraction from water-saturated coalbeds. Determining the impacts of coalbed natural gas-produced efflux water on crops is important when considering its potential use as supplemental irrigation water. We hypothesized that coalbed natural gas water, because of its high salinity and sodicity, would affect plant secondary metabolism (essential oils) and biomass accumulation. A 2-yr field study was conducted in Wyoming to investigate the effects of produced water on two traditional bioenergy feedstocks-corn ( L.) and switchgrass ( L.)-and four novel biofuel feedstock species-spearmint ( L.), Japanese cornmint ( L.), lemongrass [ (Nees ex Steud.) J.F. Watson]), and common wormwood ( L.). The four nontraditional feedstock species were chosen because they contain high-value plant chemicals that can offset production costs. Essential oil content was significantly affected by coalbed natural gas water in lemongrass and spearmint. Oil content differences between two spearmint harvests in the same year indicated that there were significant changes between the growth stage of the plant and essential oil content; the first harvest averaged 0.42 g of oil per 100 g biomass while the second harvest (harvested before flowering) yielded only 0.19 g oil per 100 g dry biomass. Results indicated that produced water can be used for short-period (2 yr) irrigation of crops. However, prolonged use of untreated produced water for irrigation would likely have deleterious long-term effects on the soil and plants unless the water was treated or diluted (mixed) with good-quality water.

Ethanol and High-Value Terpene Co-Production from Lignocellulosic Biomass of Cymbopogon flexuosus and Cymbopogon martinii.

Cymbopogon flexuosus, lemongrass, and C. martinii, palmarosa, are perennial grasses grown to produce essential oils for the fragrance industry. The objectives of this study were (1) to evaluate biomass and oil yields as a function of nitrogen and sulfur fertilization, and (2) to characterize their utility for lignocellulosic ethanol compared to Panicum virgatum (switchgrass). Mean biomass yields were 12.83 Mg lemongrass ha-1 and 15.11 Mg palmarosa ha-1 during the second harvest year resulting in theoretical biofuel yields of 2541 and 2569 L ethanol ha-1 respectively compared to reported 1749-3691 L ethanol ha-1 for switchgrass. Pretreated lemongrass yielded 198 mL ethanol (g biomass)-1 and pretreated palmarosa yielded 170 mL ethanol (g biomass)-1. Additionally, lemongrass yielded 85.7 kg essential oil ha-1 and palmarosa yielded 67.0 kg ha-1 with an estimated value of USD $857 and $1005 ha-1. These data suggest that dual-use crops such as lemongrass and palmarosa may increase the economic viability of lignocellulosic biofuels.

Ketocarotenoid Production in Soybean Seeds through Metabolic Engineering.

The pink or red ketocarotenoids, canthaxanthin and astaxanthin, are used as feed additives in the poultry and aquaculture industries as a source of egg yolk and flesh pigmentation, as farmed animals do not have access to the carotenoid sources of their wild counterparts. Because soybean is already an important component in animal feed, production of these carotenoids in soybean could be a cost-effective means of delivery. In order to characterize the ability of soybean seed to produce carotenoids, soybean cv. Jack was transformed with the crtB gene from Pantoea ananatis, which codes for phytoene synthase, an enzyme which catalyzes the first committed step in the carotenoid pathway. The crtB gene was engineered together in combinations with ketolase genes (crtW from Brevundimonas sp. strain SD212 and bkt1 from Haematococcus pluvialis) to produce ketocarotenoids; all genes were placed under the control of seed-specific promoters. HPLC results showed that canthaxanthin is present in the transgenic seeds at levels up to 52 µg/g dry weight. Transgenic seeds also accumulated other compounds in the carotenoid pathway, such as astaxanthin, lutein, ß-carotene, phytoene, α-carotene, lycopene, and ß-cryptoxanthin, whereas lutein was the only one of these detected in non-transgenic seeds. The accumulation of astaxanthin, which requires a ß-carotene hydroxylase in addition to a ß-carotene ketolase, in the transgenic seeds suggests that an endogenous soybean enzyme is able to work in combination with the ketolase transgene. Soybean seeds that accumulate ketocarotenoids could potentially be used in animal feed to reduce or eliminate the need for the costly addition of these compounds.

Morphology and ploidy level determination of Pteris vittata callus during induction and regeneration.

BACKGROUND: Morphological and ploidy changes of the arsenic hyperaccumulator, Chinese brake fern (Pteris vittata) callus tissue are described here to provide insight into fern life cycle biology and for possible biotechnology applications. Pteris vittata callus was studied using transmission and scanning electron microscopy, and flow cytometry. RESULTS: Callus induction occurred both in light and dark culture conditions from prothallus tissues, whereas rhizoid formation occurred only in dark culture conditions. Callus tissues contained two types of cells: one actively dividing and the other containing a single large vacuole undergoing exocytosis. Sporophytes regenerated from callus asynchronously form clusters of cells in a manner apparently analogous to direct organogenesis. Extracellular matrices were observed in actively-growing callus and at the base of regenerating sporophytes. Callus tissue nuclei were found to be primarily diploid at induction and throughout maintenance of cultures indicating that callus cell fate is determined at induction, which closely follows apogamous sporophyte development. Presence of a dense extracellular matrix in conjunction with sporophyte development suggests a link between the suspensor-like activity of the embryonic foot during normal fern embryo development and the suspected functions of extracellular matrices in angiosperms. CONCLUSIONS: Further investigation could lead to a better understanding of genes involved in P. vittata embryo development and apogamous sporophyte development. The methodology could be useful for in vitro propagation of rare and valuable fern germplasm.

Stable transformation of ferns using spores as targets: Pteris vittata and Ceratopteris thalictroides.

Ferns (Pteridophyta) are very important members of the plant kingdom that lag behind other taxa with regards to our understanding of their genetics, genomics, and molecular biology. We report here, to our knowledge, the first instance of stable transformation of fern with recovery of transgenic sporophytes. Spores of the arsenic hyperaccumulating fern Pteris vittata and tetraploid 'C-fern Express' (Ceratopteris thalictroides) were stably transformed by Agrobacterium tumefaciens with constructs containing the P. vittata actin promoter driving a GUSPlus reporter gene. Reporter gene expression assays were performed on multiple tissues and growth stages of gametophytes and sporophytes. Southern-blot analysis confirmed stable transgene integration in recovered sporophytes and also confirmed that no plasmid from A. tumefaciens was present in the sporophyte tissues. We recovered seven independent transformants of P. vittata and four independent C. thalictroides transgenics. Inheritance analyses using ß-glucuronidase (GUS) histochemical staining revealed that the GUS transgene was stably expressed in second generation C. thalictroides sporophytic tissues. In an independent experiment, the gusA gene that was driven by the 2× Cauliflower mosaic virus 35S promoter was bombarded into P. vittata spores using biolistics, in which putatively stable transgenic gametophytes were recovered. Transformation procedures required no tissue culture or selectable marker genes. However, we did attempt to use hygromycin selection, which was ineffective for recovering transgenic ferns. This simple stable transformation method should help facilitate functional genomics studies in ferns.

Switchgrass (Panicum virgatum L.) cell suspension cultures: Establishment, characterization, and application.

Switchgrass (Panicum virgatum L.) is a warm-season perennial grass that has received considerable attention as a potential dedicated biofuel and bioproduct feedstock. Genetic improvement of switchgrass is needed for better cellulosic ethanol production, especially to improve cellulose-to-lignin ratios. Cell suspension cultures offer an in vitro system for mutant selection, mass propagation, gene transfer, and cell biology. Toward this end, switchgrass cell suspension cultures were initiated from embryogenic callus obtained from genotype Alamo 2. They have been established and characterized with different cell type morphologies: sandy, fine milky, and ultrafine cultures. Characterization includes histological analysis using scanning electron microscopy, and utility using protoplast isolation. A high protoplast isolation rate of up to 10(6) protoplasts/1.0g of cells was achieved for the fine milky culture, whereas only a few protoplasts were isolated for the sandy and ultrafine cultures. These results indicate that switchgrass cell suspension type sizably impacts the efficiency of protoplast isolation, suggesting its significance in other applications. The establishment of different switchgrass suspension culture cell types provides the opportunity to gain insights into the versatility of the system that would further augment switchgrass biology research.

Switchgrass (Panicum virgatum L.) polyubiquitin gene (PvUbi1 and PvUbi2) promoters for use in plant transformation.

BACKGROUND: The ubiquitin protein is present in all eukaryotic cells and promoters from ubiquitin genes are good candidates to regulate the constitutive expression of transgenes in plants. Therefore, two switchgrass (Panicum virgatum L.) ubiquitin genes (PvUbi1 and PvUbi2) were cloned and characterized. Reporter constructs were produced containing the isolated 5' upstream regulatory regions of the coding sequences (i.e. PvUbi1 and PvUbi2 promoters) fused to the uidA coding region (GUS) and tested for transient and stable expression in a variety of plant species and tissues. RESULTS: PvUbi1 consists of 607 bp containing cis-acting regulatory elements, a 5' untranslated region (UTR) containing a 93 bp non-coding exon and a 1291 bp intron, and a 918 bp open reading frame (ORF) that encodes four tandem, head -to-tail ubiquitin monomer repeats followed by a 191 bp 3' UTR. PvUbi2 consists of 692 bp containing cis-acting regulatory elements, a 5' UTR containing a 97 bp non-coding exon and a 1072 bp intron, a 1146 bp ORF that encodes five tandem ubiquitin monomer repeats and a 183 bp 3' UTR. PvUbi1 and PvUbi2 were expressed in all examined switchgrass tissues as measured by qRT-PCR. Using biolistic bombardment, PvUbi1 and PvUbi2 promoters showed strong expression in switchgrass and rice callus, equaling or surpassing the expression levels of the CaMV 35S, 2x35S, ZmUbi1, and OsAct1 promoters. GUS staining following stable transformation in rice demonstrated that the PvUbi1 and PvUbi2 promoters drove expression in all examined tissues. When stably transformed into tobacco (Nicotiana tabacum), the PvUbi2+3 and PvUbi2+9 promoter fusion variants showed expression in vascular and reproductive tissues. CONCLUSIONS: The PvUbi1 and PvUbi2 promoters drive expression in switchgrass, rice and tobacco and are strong constitutive promoter candidates that will be useful in genetic transformation of monocots and dicots.

Elevational and seasonal variation in the foliar quality and arthropod community of Acer pensylvanicum.

Elevational gradients provide natural experiments for examining how variation in abiotic forces such as nutrient mineralization rates, risk of photodamge, temperature, and precipitation influence plant-insect interactions. At the Coweeta LTER site in the Southern Appalachian Mountains, we examined spatial and temporal variation in striped maple, Acer pensylvanicum, foliar quality and associated patterns in the arthropod community. Variation in herbivore densities was associated more strongly with seasonal variation in plant quality than with spatial variation in quality among three sampling sites. Leaf chewer, but not phloem feeder or arthropod predator, densities increased with elevation. Foliar quality, by our measures, decreased throughout the growing season, with decreases in nitrogen concentrations and increases in lignin concentrations. Foliar quality varied among the three sites but not systematically along the elevational gradient. We conclude that, in this system, temporal heterogeneity in plant quality is likely to be more important to insect herbivores than is spatial heterogeneity and that other factors, such as the abiotic environment and natural enemies, likely have substantial effects on herbivore density.