Charles Neal Stewart

Evaluating methods for isolating total RNA and predicting the success of sequencing phylogenetically diverse plant transcriptomes

Next-generation sequencing plays a central role in the characterization and quantification of transcriptomes. Although numerous metrics are purported to quantify the quality of RNA, there have been no large-scale empirical evaluations of the major determinants of sequencing success. We used a combination of existing and newly developed methods to isolate total RNA from 1115 samples from 695 plant species in 324 families, which represents >900 million years of phylogenetic diversity from green algae through flowering plants, including many plants of economic importance. We then sequenced 629 of these samples on Illumina GAIIx and HiSeq platforms and performed a large comparative analysis to identify predictors of RNA quality and the diversity of putative genes (scaffolds) expressed within samples. Tissue types (e.g., leaf vs. flower) varied in RNA quality, sequencing depth and the number of scaffolds. Tissue age also influenced RNA quality but not the number of scaffolds ≥1000 bp. Overall, 36% of the variation in the number of scaffolds was explained by metrics of RNA integrity (RIN score), RNA purity (OD 260/230), sequencing platform (GAIIx vs HiSeq) and the amount of total RNA used for sequencing. However, our results show that the most commonly used measures of RNA quality (e.g., RIN) are weak predictors of the number of scaffolds because Illumina sequencing is robust to variation in RNA quality. These results provide novel insight into the methods that are most important in isolating high quality RNA for sequencing and assembling plant transcriptomes. The methods and recommendations provided here could increase the efficiency and decrease the cost of RNA sequencing for individual labs and genome centers.

Multiple Polyploidy Events in the Early Radiation of Nodulating and Nonnodulating Legumes

Unresolved questions about evolution of the large and diverse legume family include the timing of polyploidy (whole-genome duplication; WGDs) relative to the origin of the major lineages within the Fabaceae and to the origin of symbiotic nitrogen fixation. Previous work has established that a WGD affects most lineages in the Papilionoideae and occurred sometime after the divergence of the papilionoid and mimosoid clades, but the exact timing has been unknown. The history of WGD has also not been established for legume lineages outside the Papilionoideae. We investigated the presence and timing of WGDs in the legumes by querying thousands of phylogenetic trees constructed from transcriptome and genome data from 20 diverse legumes and 17 outgroup species. The timing of duplications in the gene trees indicates that the papilionoid WGD occurred in the common ancestor of all papilionoids. The earliest diverging lineages of the Papilionoideae include both nodulating taxa, such as the genistoids (e.g., lupin), dalbergioids (e.g., peanut), phaseoloids (e.g., beans), and galegoids (=Hologalegina, e.g., clovers), and clades with nonnodulating taxa including Xanthocercis and Cladrastis (evaluated in this study). We also found evidence for several independent WGDs near the base of other major legume lineages, including the Mimosoideae-Cassiinae-Caesalpinieae (MCC), Detarieae, and Cercideae clades. Nodulation is found in the MCC and papilionoid clades, both of which experienced ancestral WGDs. However, there are numerous nonnodulating lineages in both clades, making it unclear whether the phylogenetic distribution of nodulation is due to independent gains or a single origin followed by multiple losses.

Genome engineering via TALENs and CRISPR/Cas9 systems: challenges and perspectives

The ability to precisely modify genome sequence and regulate gene expression patterns in a site-specific manner holds much promise in plant biotechnology. Genome-engineering technologies that enable such highly specific and efficient modification are advancing with unprecedented pace. Transcription activator-like effectors (TALEs) provide customizable DNA-binding modules designed to bind to any sequence of interest. Thus, TALEs have been used as a DNA targeting module fused to functional domains for a variety of targeted genomic and epigenomic modifications. TALE nucleases (TALENs) have been used with much success across eukaryotic species to edit genomes. Recently, clustered regularly interspaced palindromic repeats (CRISPRs) that are used as guide RNAs for Cas9 nuclease-specific digestion has been introduced as a highly efficient DNA-targeting platform for genome editing and regulation. Here, we review the discovery, development and limitations of TALENs and CRIPSR/Cas9 systems as genome-engineering platforms in plants. We discuss the current questions, potential improvements and the development of the next-generation genome-editing platforms with an emphasis on producing designer plants to address the needs of agriculture and basic plant biology.

Altered lignin biosynthesis using biotechnology to improve lignocellulosic biofuel feedstocks.

Lignocellulosic feedstocks can be converted to biofuels, which can conceivably replace a large fraction of fossil fuels currently used for transformation. However, lignin, a prominent constituent of secondary cell walls, is an impediment to the conversion of cell walls to fuel: the recalcitrance problem. Biomass pretreatment for removing lignin is the most expensive step in the production of lignocellulosic biofuels. Even though we have learned a great deal about the biosynthesis of lignin, we do not fully understand its role in plant biology, which is needed for the rational design of engineered cell walls for lignocellulosic feedstocks. This review will recapitulate our knowledge of lignin biosynthesis and discuss how lignin has been modified and the consequences for the host plant.

Evolution and spread of glyphosate resistance in Conyza canadensis in California.

Recent increases in glyphosate use in perennial crops of California, USA, are hypothesized to have led to an increase in selection and evolution of resistance to the herbicide in Conyza canadensis populations. To gain insight into the evolutionary origins and spread of resistance and to inform glyphosate resistance management strategies, we investigated the geographical distribution of glyphosate resistance in C. canadensis across and surrounding the Central Valley, its spatial relationship to groundwater protection areas (GWPA), and the genetic diversity and population structure and history using microsatellite markers. Frequencies of resistant individuals in 42 sampled populations were positively correlated with the size of GWPA within counties. Analyses of population genetic structure also supported spread of resistance in these areas. Bayesian clustering and approximate Bayesian computation (ABC) analyses revealed multiple independent origins of resistance within the Central Valley. Based on parameter estimation in the ABC analyses, resistant genotypes underwent expansion after glyphosate use began in agriculture, but many years before it was detected. Thus, diversity in weed control practices prior to herbicide regulation in GWPA probably kept resistance frequencies low. Regionally coordinated efforts to reduce seed dispersal and selection pressure are needed to manage glyphosate resistance in C. canadensis.

Identification and overexpression of gibberellin 2‐oxidase (GA2ox) in switchgrass (Panicum virgatum L.) for improved plant architecture and reduced biomass recalcitrance

Gibberellin 2-oxidases (GA2oxs) are a group of 2-oxoglutarate-dependent dioxygenases that catalyse the deactivation of bioactive GA or its precursors through 2β-hydroxylation reaction. In this study, putatively novel switchgrass C20 GA2ox genes were identified with the aim of genetically engineering switchgrass for improved architecture and reduced biomass recalcitrance for biofuel. Three C20 GA2ox genes showed differential regulation patterns among tissues including roots, seedlings and reproductive parts. Using a transgenic approach, we showed that overexpression of two C20 GA2ox genes, that is PvGA2ox5 and PvGA2ox9, resulted in characteristic GA-deficient phenotypes with dark-green leaves and modified plant architecture. The changes in plant morphology appeared to be associated with GA2ox transcript abundance. Exogenous application of GA rescued the GA-deficient phenotypes in transgenic lines. Transgenic semi-dwarf lines displayed increased tillering and reduced lignin content, and the syringyl/guaiacyl lignin monomer ratio accompanied by the reduced expression of lignin biosynthetic genes compared to nontransgenic plants. A moderate increase in the level of glucose release in these transgenic lines might be attributed to reduced biomass recalcitrance as a result of reduced lignin content and lignin composition. Our results suggest that overexpression of GA2ox genes in switchgrass is a feasible strategy to improve plant architecture and reduce biomass recalcitrance for biofuel.

Within-plant distribution and emission of sesquiterpenes from Copaifera officinalis

Copaifera officinalis, the diesel tree, is known for massive production of oleoresin, mainly composed of sesquiterpene hydrocarbons. In this study, composition of these sesquiterpenes and their concentrations in leaves, stems and roots of C. officinalis at two developmental stages, including the three-week old (TW) seedlings and two-year old (TY) trees, were determined. The leaves of TW seedlings and TY trees contained similar number of sesquiterpenes, which also had comparable concentrations. The stems of TW seedlings had higher concentrations of sesquiterpenes than those of TY trees. In contrast, the number of sesquiterpene species and their concentrations in the roots of TW seedlings were much lower than those in the roots of TY trees. Cluster analysis of sesquiterpenes estimated that there are at least four terpene synthase genes involved in the production of sesquiterpenes in C. officinalis. Because sesquiterpenes are highly volatile, emissions of sesquiterpenes from healthy and wounded TW seedlings were examined using headspace analysis. Whereas very low emission of sesquiterpenes was detected from undamaged plants, the physically injured seedlings emitted a large number of sesquiterpenes, the quality and the relative quantity of which were similar to those in leaves determined using organic extraction. The implications of our findings to the biosynthetic pathways leading to the production of sesquiterpenes as well as their biological roles in C. officinalis are discussed.

Advances in biotechnology and genomics of switchgrass

Switchgrass (Panicum virgatum L.) is a C4 perennial warm season grass indigenous to the North American tallgrass prairie. A number of its natural and agronomic traits, including adaptation to a wide geographical distribution, low nutrient requirements and production costs, high water use efficiency, high biomass potential, ease of harvesting, and potential for carbon storage, make it an attractive dedicated biomass crop for biofuel production. We believe that genetic improvements using biotechnology will be important to realize the potential of the biomass and biofuel-related uses of switchgrass. Tissue culture techniques aimed at rapid propagation of switchgrass and genetic transformation protocols have been developed. Rapid progress in genome sequencing and bioinformatics has provided efficient strategies to identify, tag, clone and manipulate many economically-important genes, including those related to higher biomass, saccharification efficiency, and lignin biosynthesis. Application of the best genetic tools should render improved switchgrass that will be more economically and environmentally sustainable as a lignocellulosic bioenergy feedstock.

From genomics to functional markers in the era of next-generation sequencing

The availability of complete genome sequences, along with other genomic resources for Arabidopsis, rice, pigeon pea, soybean and other crops, has revolutionized our understanding of the genetic make-up of plants. Next-generation DNA sequencing (NGS) has facilitated single nucleotide polymorphism discovery in plants. Functionally-characterized sequences can be identified and functional markers (FMs) for important traits can be developed at an ever-increasing ease. FMs are derived from sequence polymorphisms found in allelic variants of a functional gene. Linkage disequilibrium-based association mapping and homologous recombinants have been developed for identification of “perfect” markers for their use in crop improvement practices. Compared with many other molecular markers, FMs derived from the functionally characterized sequence genes using NGS techniques and their use provide opportunities to develop high-yielding plant genotypes resistant to various stresses at a fast pace.

Antimicrobial Activity of Yerba Mate (Ilex paraguariensis) Aqueous Extracts against Escherichia coli O157:H7 and Staphylococcus aureus

UNLABELLED Bioactive compounds from natural plant sources are becoming increasingly important to the food industry. Ilex paraguariensis is used in the preparation of a widely popular tea beverage (Yerba Mate) in the countries of Uruguay, Paraguay, Argentina, and Brazil. In this study, extracts of 4 brands of commercial tea, derived from the holly plant species, Ilex paraguariensis, were evaluated for their ability to inhibit or inactivate bacterial foodborne pathogens. The ultimate goal was to evaluate potential use of the extracts in commercial applications. Dialyzed aqueous extracts were screened for antimicrobial activity against Escherichia coli O157:H7 and Staphylococcus aureus. S. aureus was found to be the more sensitive to extracts than E. coli O157:H7. Minimum bactericidal concentrations (MBCs) were determined to be approximately 150 to 800 μg/mL and 25 to 50 μg/mL against E. coli O157:H7 and S. aureus, respectively. A Uruguayan brand had reduced activity against E. coli O157:H7 compared to the Argentinean brands tested. It was concluded that Yerba Mate could be used as a potential antimicrobial in foods and beverages against these pathogenic bacteria. PRACTICAL APPLICATION Soluble extracts from Yerba Mate are natural antimicrobials that can be incorporated into food products to achieve longer shelf life.