The final piece of the Triangle of U: Evolution of the tetraploid Brassica carinata genome.

Ethiopian mustard (Brassica carinata) is an ancient crop with remarkable stress resilience and a desirable seed fatty acid profile for biofuel uses. Brassica carinata is one of six Brassica species that share three major genomes from three diploid species (AA, BB, and CC) that spontaneously hybridized in a pairwise manner to form three allotetraploid species (AABB, AACC, and BBCC). Of the genomes of these species, that of B. carinata is the least understood. Here, we report a chromosome scale 1.31-Gbp genome assembly with 156.9-fold sequencing coverage for B. carinata, completing the reference genomes comprising the classic Triangle of U, a classical theory of the evolutionary relationships among these six species. Our assembly provides insights into the hybridization event that led to the current B. carinata genome and the genomic features that gave rise to the superior agronomic traits of B. carinata. Notably, we identified an expansion of transcription factor networks and agronomically important gene families. Completion of the Triangle of U comparative genomics platform has allowed us to examine the dynamics of polyploid evolution and the role of subgenome dominance in the domestication and continuing agronomic improvement of B. carinata and other Brassica species.

Structural diversity, biosynthesis, and function of plant falcarin-type polyacetylenic lipids.

The polyacetylenic lipids falcarinol, falcarindiol, and associated derivatives, termed falcarins, have a widespread taxonomical distribution in the plant kingdom and have received increasing interest for their demonstrated health-promoting properties as anti-cancer and anti-inflammatory agents. These fatty acid-derived compounds are also linked to plant pathogen resistance through their potent antimicrobial properties. Falcarin-type polyacetylenes, which contain two conjugated triple bonds, are derived from structural modifications of the common fatty acid oleic acid. In the past half century, much progress has been made in understanding the structural diversity of falcarins in the plant kingdom, whereas limited progress has been made on elucidating falcarin function in plant-pathogen interactions. More recently, an understanding of the biosynthetic machinery underlying falcarin biosynthesis has emerged. This review provides a concise summary of the current state of knowledge on falcarin structural diversity, biosynthesis, and plant defense properties. We also present major unanswered questions about falcarin biosynthesis and function.

Plant tissue succulence engineering improves water-use efficiency, water-deficit stress attenuation and salinity tolerance in Arabidopsis.

Tissue succulence (ratio of tissue water/leaf area or dry mass) or the ability to store water within living tissues is among the most successful adaptations to drought in the plant kingdom. This taxonomically widespread adaptation helps plants avoid the damaging effects of drought, and is often associated with the occupancy of epiphytic, epilithic, semi-arid and arid environments. Tissue succulence was engineered in Arabidopsis thaliana by overexpression of a codon-optimized helix-loop-helix transcription factor (VvCEB1opt ) from wine grape involved in the cell expansion phase of berry development. VvCEB1opt -overexpressing lines displayed significant increases in cell size, succulence and decreased intercellular air space. VvCEB1opt -overexpressing lines showed increased instantaneous and integrated water-use efficiency (WUE) due to reduced stomatal conductance caused by reduced stomatal aperture and density resulting in increased attenuation of water-deficit stress. VvCEB1opt -overexpressing lines also showed increased salinity tolerance due to reduced salinity uptake and dilution of internal Na+ and Cl- as well as other ions. Alterations in transporter activities were further suggested by media and apoplastic acidification, hygromycin B tolerance and changes in relative transcript abundance patterns of various transporters with known functions in salinity tolerance. Engineered tissue succulence might provide an effective strategy for improving WUE, drought avoidance or attenuation, salinity tolerance, and for crassulacean acid metabolism biodesign.

Identification of Genes Encoding Enzymes Catalyzing the Early Steps of Carrot Polyacetylene Biosynthesis.

Polyacetylenic lipids accumulate in various Apiaceae species after pathogen attack, suggesting that these compounds are naturally occurring pesticides and potentially valuable resources for crop improvement. These compounds also promote human health and slow tumor growth. Even though polyacetylenic lipids were discovered decades ago, the biosynthetic pathway underlying their production is largely unknown. To begin filling this gap and ultimately enable polyacetylene engineering, we studied polyacetylenes and their biosynthesis in the major Apiaceae crop carrot (Daucus carota subsp. sativus). Using gas chromatography and mass spectrometry, we identified three known polyacetylenes and assigned provisional structures to two novel polyacetylenes. We also quantified these compounds in carrot leaf, petiole, root xylem, root phloem, and root periderm extracts. Falcarindiol and falcarinol predominated and accumulated primarily in the root periderm. Since the multiple double and triple carbon-carbon bonds that distinguish polyacetylenes from ubiquitous fatty acids are often introduced by Δ12 oleic acid desaturase (FAD2)-type enzymes, we mined the carrot genome for FAD2 genes. We identified a FAD2 family with an unprecedented 24 members and analyzed public, tissue-specific carrot RNA-Seq data to identify coexpressed members with root periderm-enhanced expression. Six candidate genes were heterologously expressed individually and in combination in yeast and Arabidopsis (Arabidopsis thaliana), resulting in the identification of one canonical FAD2 that converts oleic to linoleic acid, three divergent FAD2-like acetylenases that convert linoleic into crepenynic acid, and two bifunctional FAD2s with Δ12 and Δ14 desaturase activity that convert crepenynic into the further desaturated dehydrocrepenynic acid, a polyacetylene pathway intermediate. These genes can now be used as a basis for discovering other steps of falcarin-type polyacetylene biosynthesis, to modulate polyacetylene levels in plants, and to test the in planta function of these molecules.

A roadmap for research on crassulacean acid metabolism (CAM) to enhance sustainable food and bioenergy production in a hotter, drier world.

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that features nocturnal CO2 uptake, facilitates increased water-use efficiency (WUE), and enables CAM plants to inhabit water-limited environments such as semi-arid deserts or seasonally dry forests. Human population growth and global climate change now present challenges for agricultural production systems to increase food, feed, forage, fiber, and fuel production. One approach to meet these challenges is to increase reliance on CAM crops, such as Agave and Opuntia, for biomass production on semi-arid, abandoned, marginal, or degraded agricultural lands. Major research efforts are now underway to assess the productivity of CAM crop species and to harness the WUE of CAM by engineering this pathway into existing food, feed, and bioenergy crops. An improved understanding of CAM has potential for high returns on research investment. To exploit the potential of CAM crops and CAM bioengineering, it will be necessary to elucidate the evolution, genomic features, and regulatory mechanisms of CAM. Field trials and predictive models will be required to assess the productivity of CAM crops, while new synthetic biology approaches need to be developed for CAM engineering. Infrastructure will be needed for CAM model systems, field trials, mutant collections, and data management.

What happens when the lights are left on? Transcriptomic and phenotypic habituation to light pollution.

Artificial light at night (ALAN) is a ubiquitous pollutant worldwide. Exposure can induce immediate behavioral and physiological changes in animals, sometimes leading to severe health consequences. Nevertheless, many organisms persist in light-polluted environments and may have mechanisms of habituating, reducing responses to repeated exposure over time, but this has yet to be tested experimentally. Here, we tested whether zebra finches (Taeniopygia guttata) can habituate to dim (0.3 lux) ALAN, measuring behavior, physiology (oxidative stress and telomere attrition), and gene expression in a repeated measures design, over 6 months. We present evidence of tolerance to chronic exposure, persistent behavioral responses lasting 8 weeks post-exposure, and attenuation of responses to re-exposure. Oxidative stress decreased under chronic ALAN. Changes in the blood transcriptome revealed unique responses to past exposure and re-exposure. Results demonstrate organismal resilience to chronic stressors and shed light on the capacity of birds to persist in an increasingly light-polluted world.

PLANEX: the plant co-expression database.

BACKGROUND: The PLAnt co-EXpression database (PLANEX) is a new internet-based database for plant gene analysis. PLANEX (http://planex.plantbioinformatics.org) contains publicly available GeneChip data obtained from the Gene Expression Omnibus (GEO) of the National Center for Biotechnology Information (NCBI). PLANEX is a genome-wide co-expression database, which allows for the functional identification of genes from a wide variety of experimental designs. It can be used for the characterization of genes for functional identification and analysis of a gene's dependency among other genes. Gene co-expression databases have been developed for other species, but gene co-expression information for plants is currently limited. DESCRIPTION: We constructed PLANEX as a list of co-expressed genes and functional annotations for Arabidopsis thaliana, Glycine max, Hordeum vulgare, Oryza sativa, Solanum lycopersicum, Triticum aestivum, Vitis vinifera and Zea mays. PLANEX reports Pearson's correlation coefficients (PCCs; r-values) that distribute from a gene of interest for a given microarray platform set corresponding to a particular organism. To support PCCs, PLANEX performs an enrichment test of Gene Ontology terms and Cohen's Kappa value to compare functional similarity for all genes in the co-expression database. PLANEX draws a cluster network with co-expressed genes, which is estimated using the k-mean method. To construct PLANEX, a variety of datasets were interpreted by the IBM supercomputer Advanced Interactive eXecutive (AIX) in a supercomputing center. CONCLUSION: PLANEX provides a correlation database, a cluster network and an interpretation of enrichment test results for eight plant species. A typical co-expressed gene generates lists of co-expression data that contain hundreds of genes of interest for enrichment analysis. Also, co-expressed genes can be identified and cataloged in terms of comparative genomics by using the 'Co-expression gene compare' feature. This type of analysis will help interpret experimental data and determine whether there is a common term among genes of interest.

Transcriptional network analysis of the tryptophan-accumulating rice mutant during grain filling.

In a previous study, we selected a high tryptophan (Trp)-accumulating rice (Oryza sativa L.) mutant line by in vitro mutagenesis using gamma rays. To obtain detailed information about the Trp biosynthetic pathway during the grain-filling in rice, we investigated the gene expression profiles in the wild-type (cv. Dongan) and the high-level Trp-accumulating mutant line (MRVII-33) at five different grain-filling stages using microarray analysis. The mutant line showed approximately 6.3-fold higher Trp content and 2.3-fold higher amino acids compared with the original cultivar at the final stage (stage V). The intensity of gene expression was analyzed and compared between the wild-type and mutant line at each of the five grain-filling stages using the Rice 4 × 44K oligo DNA microarray. Among the five stages, stage III showed the highest gene expression changes for both up- and down-regulated genes. Among the Trp biosynthesis-related genes, trpG showed high expression in the mutant line during stages I to IV and trpE showed higher at stage III. Gene clustering was performed based on the genes of KEGG's amino acid metabolism, and a total of 276 genes related to amino acid metabolism were placed into three clusters. The functional annotation enrichment analysis of the genes classified into the three clusters was also conducted using ClueGO. It was found that cluster 3 uniquely included biological processes related to aromatic amino acid metabolism. These results suggest that gene analysis based on microarray data is useful for elucidating the biological mechanisms of Trp accumulation in high Trp-accumulating mutants at each of the grain-filling stages.

Characterization of a microbial consortium with potential for biological degradation of cactus pear biomass for biofuel production.

Cactus pear (Opuntia ficus-indica) is a crassulacean acid metabolism (CAM) species that serves as a food, feed, and bioenergy crop. O. ficus-indica is an attractive alternative biofuel feedstock due to its low water demand and high biomass productivity. Current ethanol yields from O. ficus-indica are not commercially viable due to low concentrations of released fermentable carbohydrates. Axenic strains of bacteria and fungi were isolated and characterized from a soil microbial community consortium that effectively degrades cladodes into soluble components. The consortium consisted of species representing 14 genera of eubacteria and four genera of fungi. The digestion efficiency of each axenic isolate was evaluated by measuring the release of soluble material after aerobic digestion of cladodes and direct measurement of cellulase and pectinase activities in the culture supernatants. Pectobacterium cacticida was the most effective eubacterial species identified for degrading cladodes among all isolates evaluated. Thus, P. cacticida holds great promise for increasing the release of fermentable sugars and improving overall ethanol yields.

A gene family encoding RING finger proteins in rice: their expansion, expression diversity, and co-expressed genes.

The proteins harboring RING finger motif(s) have been shown to mediate protein-protein interactions that are relevant to a variety of cellular processes. In an effort to elucidate the evolutionary dynamics of the rice RING finger protein family, we have attempted to determine their genomic locations, expression diversity, and co-expressed genes via in silico analysis and semi-quantitative RT-PCR. A total of 425 retrieved genes appear to be distributed over all 12 of the chromosomes of rice with different distributions, and are reflective of the evolutionary dynamics of the rice genome. A genome-wide dataset harboring 155 gene expression omnibus sample plates evidenced some degree of differential evolutionary fates between members of RING-H2 and RING-HC types. Additionally, responses to abiotic stresses, such as salinity and drought, demonstrated that some degree of expression diversity existed between members of the RING finger protein genes. Interestingly, we determined that one RING-H2 finger protein gene (Os04g51400) manifested striking differences in expression patterns in response to abiotic stresses between leaf and culm-node tissues, further revealing responses highly similar to the majority of randomly selected co-expressed genes. The gene network of genes co-expressed with Os04g51400 may suggest some role in the salt response of the gene. These findings may shed further light on the evolutionary dynamics and molecular functional diversity of these proteins in complex cellular regulations.

Expressional diversity of wheat nsLTP genes: evidence of subfunctionalization via cis-regulatory divergence.

Previously, the wheat non-specific lipid transfer proteins (TaLTP), members of a small multigene family, were reported to evidence a complex pattern of expression regulation. In order to assess further the expression diversity of the TaLTP genes, we have attempted to evaluate their expression profiles in responses to abiotic stresses, using semi-quantitative RT-PCR. The expression profiles generated herein revealed that the TaLTP genes in group A evidenced highly similar responses against abiotic stresses, whereas differential expression patterns among genes in each group were also observed. A total of seven promoters were fused to a GUS reporter gene and the recombinants were introduced into Arabidopsis, while three promoters evidenced non-detectible GUS activity. The promoters of TaLTP1, TaLTP7, and TaLTP10 included in group A drove strong expressions during plant development with overlapping patterns, in large part, but also exhibited distinct expression pattern, thereby suggesting subfunctionalization processing over evolutionary time. However, only trace expression in cotyledons, young emerged leaves, and epidermal cell layers of flower ovaries was driven by the promoter of TaLTP3 of group B. These results indicate that their distinct physiological functions appear to be accomplished by a subfunctionalization process involving degenerative mutations in regulatory regions.

Biosystems Design to Accelerate C3-to-CAM Progression.

Global demand for food and bioenergy production has increased rapidly, while the area of arable land has been declining for decades due to damage caused by erosion, pollution, sea level rise, urban development, soil salinization, and water scarcity driven by global climate change. In order to overcome this conflict, there is an urgent need to adapt conventional agriculture to water-limited and hotter conditions with plant crop systems that display higher water-use efficiency (WUE). Crassulacean acid metabolism (CAM) species have substantially higher WUE than species performing C3 or C4 photosynthesis. CAM plants are derived from C3 photosynthesis ancestors. However, it is extremely unlikely that the C3 or C4 crop plants would evolve rapidly into CAM photosynthesis without human intervention. Currently, there is growing interest in improving WUE through transferring CAM into C3 crops. However, engineering a major metabolic plant pathway, like CAM, is challenging and requires a comprehensive deep understanding of the enzymatic reactions and regulatory networks in both C3 and CAM photosynthesis, as well as overcoming physiometabolic limitations such as diurnal stomatal regulation. Recent advances in CAM evolutionary genomics research, genome editing, and synthetic biology have increased the likelihood of successful acceleration of C3-to-CAM progression. Here, we first summarize the systems biology-level understanding of the molecular processes in the CAM pathway. Then, we review the principles of CAM engineering in an evolutionary context. Lastly, we discuss the technical approaches to accelerate the C3-to-CAM transition in plants using synthetic biology toolboxes.

Expression diversity and evolutionary dynamics of rice duplicate genes.

Duplicate genes are believed to be a major source of new gene functions over evolutionary time. In order to evaluate the evolutionary dynamics of rice duplicate genes, formed principally by paleoployploidization prior to the speciation of the Poaceae family, we have employed a public microarray dataset including 155 gene expression omnibus sample plates and bioinformatics tools. At least 57.4% of old approximately 70 million years ago (MYA) duplicate gene pairs exhibit divergences in expression over the given experimental set, whereas at least 50.9% of young approximately 7.7-MYA duplicate gene pairs were shown to be divergent. When grouping the rice duplicate genes according to functional categories, we noted a striking and significant enrichment of divergent duplicate metabolism-associated genes, as compared to that observed in non-divergent duplicate genes. While both non-synonymous substitution (Ka) and synonymous substitution (Ks) values between non- and divergent duplicate gene pairs evidenced significant differences, the Ka/Ks values between them exhibited no significant differences. Interestingly, the average numbers of conserved motifs of the duplicate gene pairs revealed a pattern of decline along with an increase in expression diversity, partially supporting the subfunctionalization model with degenerative complementation in regulatory motifs. Duplicate gene pairs with high local similarity (HLS) segments, which might be formed via conversion between rice paleologs, evidenced higher expression correlations than were observed in the gene pairs without the HLS segments; this probably resulted in an increased likelihood of gene conversion in promoters of the gene pairs harboring HLS segments. More than 30% [corrected] of the rice gene families exhibited similar high expression diversity between members as compared to that of randomly selected gene pairs. These findings are likely reflective of the evolutionary dynamics of rice duplicate genes for gene retention.

Laying the Foundation for Crassulacean Acid Metabolism (CAM) Biodesign: Expression of the C4 Metabolism Cycle Genes of CAM in Arabidopsis.

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that exploits a temporal CO2 pump with nocturnal CO2 uptake and concentration to reduce photorespiration, improve water-use efficiency (WUE), and optimize the adaptability of plants to hotter and drier climates. Introducing the CAM photosynthetic machinery into C3 (or C4) photosynthesis plants (CAM Biodesign) represents a potentially breakthrough strategy for improving WUE while maintaining high productivity. To optimize the success of CAM Biodesign approaches, the functional analysis of individual C4 metabolism cycle genes is necessary to identify the essential genes for robust CAM pathway introduction. Here, we isolated and analyzed the subcellular localizations of 13 enzymes and regulatory proteins of the C4 metabolism cycle of CAM from the common ice plant in stably transformed Arabidopsis thaliana. Six components of the carboxylation module were analyzed including beta-carbonic anhydrase (McBCA2), phosphoenolpyruvate carboxylase (McPEPC1), phosphoenolpyruvate carboxylase kinase (McPPCK1), NAD-dependent malate dehydrogenase (McNAD-MDH1, McNAD-MDH2), and NADP-dependent malate dehydrogenase (McNADP-MDH1). In addition, seven components of the decarboxylation module were analyzed including NAD-dependent malic enzyme (McNAD-ME1, McNAD-ME2), NADP-dependent malic enzyme (McNADP-ME1, NADP-ME2), pyruvate, orthophosphate dikinase (McPPDK), pyruvate, orthophosphate dikinase-regulatory protein (McPPDK-RP), and phosphoenolpyruvate carboxykinase (McPEPCK). Ectopic overexpression of most C4-metabolism cycle components resulted in increased rosette diameter, leaf area, and leaf fresh weight of A. thaliana except for McNADP-MDH1, McPPDK-RP, and McPEPCK. Overexpression of most carboxylation module components resulted in increased stomatal conductance and dawn/dusk titratable acidity (TA) as an indirect measure of organic acid (mainly malate) accumulation in A. thaliana. In contrast, overexpression of the decarboxylating malic enzymes reduced stomatal conductance and TA. This comprehensive study provides fundamental insights into the relative functional contributions of each of the individual components of the core C4-metabolism cycle of CAM and represents a critical first step in laying the foundation for CAM Biodesign.

Divergence of genes encoding non-specific lipid transfer proteins in the poaceae family.

The genes encoding non-specific lipid transfer proteins (nsLTPs), members of a small multigene family, show a complex pattern of expressional regulation, suggesting that some diversification may have resulted from changes in their expression after duplication. In this study, the evolution of nsLTP genes within the Poaceae family was characterized via a survey of the pseudogenes and unigenes encoding the nsLTP in rice pseudomolecules and the NCBI unigene database. nsLTP-rich regions were detected in the distal portions of rice chromosomes 11 and 12; these may have resulted from the most recent large segmental duplication in the rice genome. Two independent tandem duplications were shown to occur within the nsLTP-rich regions of rice. The genomic distribution of the nsLTP genes in the rice genome differs from that in wheat. This may be attributed to gene migration, chromosomal rearrangement, and/or differential gene loss. The genomic distribution pattern of nsLTP genes in the Poaceae family points to the existence of some differences among cereal nsLTP genes, all of which diverged from an ancient gene. The unigenes encoding nsLTPs in each cereal species are clustered into five groups. The somewhat different distribution of nsLTP-encoding EST clones between the groups across cereal species imply that independent duplication(s) followed by subfunctionalization (and/or neofunctionalization) of the nsLTP gene family in each species occurred during speciation.

Divide and Conquer (DC) BLAST: fast and easy BLAST execution within HPC environments.

Bioinformatics is currently faced with very large-scale data sets that lead to computational jobs, especially sequence similarity searches, that can take absurdly long times to run. For example, the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST and BLAST+) suite, which is by far the most widely used tool for rapid similarity searching among nucleic acid or amino acid sequences, is highly central processing unit (CPU) intensive. While the BLAST suite of programs perform searches very rapidly, they have the potential to be accelerated. In recent years, distributed computing environments have become more widely accessible and used due to the increasing availability of high-performance computing (HPC) systems. Therefore, simple solutions for data parallelization are needed to expedite BLAST and other sequence analysis tools. However, existing software for parallel sequence similarity searches often requires extensive computational experience and skill on the part of the user. In order to accelerate BLAST and other sequence analysis tools, Divide and Conquer BLAST (DCBLAST) was developed to perform NCBI BLAST searches within a cluster, grid, or HPC environment by using a query sequence distribution approach. Scaling from one (1) to 256 CPU cores resulted in significant improvements in processing speed. Thus, DCBLAST dramatically accelerates the execution of BLAST searches using a simple, accessible, robust, and parallel approach. DCBLAST works across multiple nodes automatically and it overcomes the speed limitation of single-node BLAST programs. DCBLAST can be used on any HPC system, can take advantage of hundreds of nodes, and has no output limitations. This freely available tool simplifies distributed computation pipelines to facilitate the rapid discovery of sequence similarities between very large data sets.

Evolution of non-specific lipid transfer protein (nsLTP) genes in the Poaceae family: their duplication and diversity.

Previously, the genes encoding non-specific lipid transfer proteins (nsLTPs) of the Poaceae family appear to evidence different genomic distribution and somewhat different shares of EST clones, which is suggestive of independent duplication(s) followed by functional diversity. To further evaluate the evolutionary fate of the Poaceae nsLTP genes, we have identified Ka/Ks values, conserved, mutated or lost cis-regulatory elements, responses to several elicitors, genome-wide expression profiles, and nsLTP gene-coexpression networks of both (or either) wheat and rice. The Ka/Ks values within each group and between groups appeared to be similar, but not identical, in both species. The conserved cis-regulatory elements, e.g. the RY repeat (CATGCA) element related to ABA regulation in group A, might be reflected in some degree of long-term conservation in transcriptional regulation post-dating speciation. In group A, wheat nsLTP genes, with the exception of TaLTP4, evidenced responses similar to those of plant elicitors; however, the rice nsLTP genes evidenced differences in expression profiles, even though the genes of both species have undergone purifying selection, thereby suggesting their independent functional diversity. The expression profiles of rice nsLTP genes with a microarray dataset of 155 gene expression omnibus sample (GSM) plates suggest that subfunctionalization is not the sole mechanism inherent to the evolutionary history of nsLTP genes but may, rather, function in concert with other mechanism(s). As inferred by the nsLTP gene-coexpression networks, the functional diversity of nsLTP genes appears not to be randomized, but rather to be specialized in the direction of specific biological processes over evolutionary time.