Shahjahan Ali

The genome of the extremophile crucifer Thellungiella parvula

Thellungiella parvula is related to Arabidopsis thaliana and is endemic to saline, resource-poor habitats, making it a model for the evolution of plant adaptation to extreme environments. Here we present the draft genome for this extremophile species. Exclusively by next generation sequencing, we obtained the de novo assembled genome in 1,496 gap-free contigs, closely approximating the estimated genome size of 140 Mb. We anchored these contigs to seven pseudo chromosomes without the use of maps. We show that short reads can be assembled to a near-complete chromosome level for a eukaryotic species lacking prior genetic information. The sequence identifies a number of tandem duplications that, by the nature of the duplicated genes, suggest a possible basis for T. parvulas extremophile lifestyle. Our results provide essential background for developing genomically influenced testable hypotheses for the evolution of environmental stress tolerance.

Dynamic regulation of genome-wide pre-mRNA splicing and stress tolerance by the Sm-like protein LSm5 in Arabidopsis

BackgroundSm-like proteins are highly conserved proteins that form the core of the U6 ribonucleoprotein and function in several mRNA metabolism processes, including pre-mRNA splicing. Despite their wide occurrence in all eukaryotes, little is known about the roles of Sm-like proteins in the regulation of splicing.ResultsHere, through comprehensive transcriptome analyses, we demonstrate that depletion of the Arabidopsis supersensitive to abscisic acid and drought 1 gene (SAD1), which encodes Sm-like protein 5 (LSm5), promotes an inaccurate selection of splice sites that leads to a genome-wide increase in alternative splicing. In contrast, overexpression of SAD1 strengthens the precision of splice-site recognition and globally inhibits alternative splicing. Further, SAD1 modulates the splicing of stress-responsive genes, particularly under salt-stress conditions. Finally, we find that overexpression of SAD1 in Arabidopsis improves salt tolerance in transgenic plants, which correlates with an increase in splicing accuracy and efficiency for stress-responsive genes.ConclusionsWe conclude that SAD1 dynamically controls splicing efficiency and splice-site recognition in Arabidopsis, and propose that this may contribute to SAD1-mediated stress tolerance through the metabolism of transcripts expressed from stress-responsive genes. Our study not only provides novel insights into the function of Sm-like proteins in splicing, but also uncovers new means to improve splicing efficiency and to enhance stress tolerance in a higher eukaryote.

Chromerid genomes reveal the evolutionary path from photosynthetic algae to obligate intracellular parasites

The eukaryotic phylum Apicomplexa encompasses thousands of obligate intracellular parasites of humans and animals with immense socio-economic and health impacts. We sequenced nuclear genomes of Chromera velia and Vitrella brassicaformis, free-living non-parasitic photosynthetic algae closely related to apicomplexans. Proteins from key metabolic pathways and from the endomembrane trafficking systems associated with a free-living lifestyle have been progressively and non-randomly lost during adaptation to parasitism. The free-living ancestor contained a broad repertoire of genes many of which were repurposed for parasitic processes, such as extracellular proteins, components of a motility apparatus, and DNA- and RNA-binding protein families. Based on transcriptome analyses across 36 environmental conditions, Chromera orthologs of apicomplexan invasion-related motility genes were co-regulated with genes encoding the flagellar apparatus, supporting the functional contribution of flagella to the evolution of invasion machinery. This study provides insights into how obligate parasites with diverse life strategies arose from a once free-living phototrophic marine alga. DOI: http://dx.doi.org/10.7554/eLife.06974.001

Genome structures and halophyte-specific gene expression of the extremophile Thellungiella parvula in comparison with Thellungiella salsuginea (Thellungiella halophila) and Arabidopsis.

The genome of Thellungiella parvula, a halophytic relative of Arabidopsis (Arabidopsis thaliana), is being assembled using Roche-454 sequencing. Analyses of a 10-Mb scaffold revealed synteny with Arabidopsis, with recombination and inversion and an uneven distribution of repeat sequences. T. parvula genome structure and DNA sequences were compared with orthologous regions from Arabidopsis and publicly available bacterial artificial chromosome sequences from Thellungiella salsuginea (previously Thellungiella halophila). The three-way comparison of sequences, from one abiotic stress-sensitive species and two tolerant species, revealed extensive sequence conservation and microcolinearity, but grouping Thellungiella species separately from Arabidopsis. However, the T. parvula segments are distinguished from their T. salsuginea counterparts by a pronounced paucity of repeat sequences, resulting in a 30% shorter DNA segment with essentially the same gene content in T. parvula. Among the genes is SALT OVERLY SENSITIVE1 (SOS1), a sodium/proton antiporter, which represents an essential component of plant salinity stress tolerance. Although the SOS1 coding region is highly conserved among all three species, the promoter regions show conservation only between the two Thellungiella species. Comparative transcript analyses revealed higher levels of basal as well as salt-induced SOS1 expression in both Thellungiella species as compared with Arabidopsis. The Thellungiella species and other halophytes share conserved pyrimidine-rich 5′ untranslated region proximal regions of SOS1 that are missing in Arabidopsis. Completion of the genome structure of T. parvula is expected to highlight distinctive genetic elements underlying the extremophile lifestyle of this species.

Genome-wide analysis of alternative splicing of pre-mRNA under salt stress in Arabidopsis.

BackgroundAlternative splicing (AS) of precursor mRNA (pre-mRNA) is an important gene regulation process that potentially regulates many physiological processes in plants, including the response to abiotic stresses such as salt stress.ResultsTo analyze global changes in AS under salt stress, we obtained high-coverage (~200 times) RNA sequencing data from Arabidopsis thaliana seedlings that were treated with different concentrations of NaCl. We detected that ~49% of all intron-containing genes were alternatively spliced under salt stress, 10% of which experienced significant differential alternative splicing (DAS). Furthermore, AS increased significantly under salt stress compared with under unstressed conditions. We demonstrated that most DAS genes were not differentially regulated by salt stress, suggesting that AS may represent an independent layer of gene regulation in response to stress. Our analysis of functional categories suggested that DAS genes were associated with specific functional pathways, such as the pathways for the responses to stresses and RNA splicing. We revealed that serine/arginine-rich (SR) splicing factors were frequently and specifically regulated in AS under salt stresses, suggesting a complex loop in AS regulation for stress adaptation. We also showed that alternative splicing site selection (SS) occurred most frequently at 4 nucleotides upstream or downstream of the dominant sites and that exon skipping tended to link with alternative SS.ConclusionsOur study provided a comprehensive view of AS under salt stress and revealed novel insights into the potential roles of AS in plant response to salt stress.

Arabidopsis cytochrome P450s through the looking glass: a window on plant biochemistry

Annotation of the genome sequence of Arabidopsis thaliana has identified a diverse array of 245 full-length cytochrome P450 monooxygenase (P450) genes whose known functions span the synthetic gamut from critical structural components (phenylpropanoids, fatty acids, sterols) to signaling molecules (oxylipins, brassinosteroids, abscisic acid, gibberellic acid) and defense compounds (alkaloids, terpenes, coumarins). Numerous others in this collection mediate functions that are now being addressed using microarray and oligoarray technologies, molecular modeling, heterologous expression and insertional mutageneses. Profilings of their constitutive and inducible transcript levels have begun to cluster P450s that are likely to mediate tissue-specific and stress-specific monooxygenations. With proper appreciation of the high identities that exist among some of the most recently duplicated P450 sequences, these studies have begun to differentiate P450s with early response functions leading to production of stress signaling molecules and late response functions leading to the synthesis of protective compounds. Further functional analyses of these P450 sequences with perspectives on their response profiles rely on a variety of theoretical modeling and experimental approaches that can ultimately be tied to the transcriptional profiles and genetic mutants. This review surveys historical and evolutionary aspects of P450 studies, expression variations among Arabidopsis P450 loci, catalytic site regions critical for substrate recognition and, finally, genetic mutations/disruptions that can ultimately tie biochemical reactions to physiological functions in a manner not yet possible in most other organisms.

Dissolved Organic Carbon Influences Microbial Community Composition and Diversity in Managed Aquifer Recharge Systems

ABSTRACT This study explores microbial community structure in managed aquifer recharge (MAR) systems across both laboratory and field scales. Two field sites, the Taif River (Taif, Saudi Arabia) and South Platte River (Colorado), were selected as geographically distinct MAR systems. Samples derived from unsaturated riverbed, saturated-shallow-infiltration (depth, 1 to 2 cm), and intermediate-infiltration (depth, 10 to 50 cm) zones were collected. Complementary laboratory-scale sediment columns representing low (0.6 mg/liter) and moderate (5 mg/liter) dissolved organic carbon (DOC) concentrations were used to further query the influence of DOC and depth on microbial assemblages. Microbial density was positively correlated with the DOC concentration, while diversity was negatively correlated at both the laboratory and field scales. Microbial communities derived from analogous sampling zones in each river were not phylogenetically significantly different on phylum, class, genus, and species levels, as determined by 16S rRNA gene pyrosequencing, suggesting that geography and season exerted less sway than aqueous geochemical properties. When field-scale communities derived from the Taif and South Platte River sediments were grouped together, principal coordinate analysis revealed distinct clusters with regard to the three sample zones (unsaturated, shallow, and intermediate saturated) and, further, with respect to DOC concentration. An analogous trend as a function of depth and corresponding DOC loss was observed in column studies. Canonical correspondence analysis suggests that microbial classes Betaproteobacteria and Gammaproteobacteria are positively correlated with DOC concentration. Our combined analyses at both the laboratory and field scales suggest that DOC may exert a strong influence on microbial community composition and diversity in MAR saturated zones.

Split Photosystem Protein, Linear-Mapping Topology, and Growth of Structural Complexity in the Plastid Genome of Chromera velia

The canonical photosynthetic plastid genomes consist of a single circular-mapping chromosome that encodes a highly conserved protein core, involved in photosynthesis and ATP generation. Here, we demonstrate that the plastid genome of the photosynthetic relative of apicomplexans, Chromera velia, departs from this view in several unique ways. Core photosynthesis proteins PsaA and AtpB have been broken into two fragments, which we show are independently transcribed, oligoU-tailed, translated, and assembled into functional photosystem I and ATP synthase complexes. Genome-wide transcription profiles support expression of many other highly modified proteins, including several that contain extensions amounting to hundreds of amino acids in length. Canonical gene clusters and operons have been fragmented and reshuffled into novel putative transcriptional units. Massive genomic coverage by paired-end reads, coupled with pulsed-field gel electrophoresis and polymerase chain reaction, consistently indicate that the C. velia plastid genome is linear-mapping, a unique state among all plastids. Abundant intragenomic duplication probably mediated by recombination can explain protein splits, extensions, and genome linearization and is perhaps the key driving force behind the many features that defy the conventional ways of plastid genome architecture and function.

Expression profiles of differentially regulated genes during the early stages of apple flower infection with Erwinia amylovora

To identify genes involved in the response to the fire blight pathogen Erwinia amylovora in apple (Malus×domestica), expression profiles were investigated using an apple oligo (70-mer) array representing 40, 000 genes. Blossoms of a fire blight-susceptible apple cultivar Gala were collected from trees growing in the orchard, placed on a tray in the laboratory, and spray-inoculated with a suspension of E. amylovora at a concentration of 108 cfu ml−1. Uninoculated detached flowers served as controls at each time point. Expression profiles were captured at three different time points post-inoculation at 2, 8, and 24 h, together with those at 0 h (uninoculated). A total of about 3500 genes were found to be significantly modulated in response to at least one of the three time points. Among those, a total of 770, 855, and 1002 genes were up-regulated, by 2-fold, at 2, 8, and 24 h following inoculation, respectively; while, 748, 1024, and 1455 genes were down-regulated, by 2-fold, at 2, 8, and 24 h following inoculation, respectively. Over the three time points post-inoculation, 365 genes were commonly up-regulated and 374 genes were commonly down-regulated. Both sets of genes were classified based on their functional categories. The majority of up-regulated genes were involved in metabolism, signal transduction, signalling, transport, and stress response. A number of transcripts encoding proteins/enzymes known to be up-regulated under particular biotic and abiotic stress were also up-regulated following E. amylovora treatment. Those up- or down-regulated genes encode transcription factors, signaling components, defense-related, transporter, and metabolism, all of which have been associated with disease responses in Arabidopsis and rice, suggesting similar response pathways are involved in apple blossoms.

Untranslated Regions from C4 Amaranth AhRbcS1 mRNAs Confer Translational Enhancement and Preferential Bundle Sheath Cell Expression in Transgenic C4 Flaveria bidentis

Many aspects of photosynthetic gene expression are posttranscriptionally regulated in C4 plants. To determine if RbcS mRNA untranslated regions (UTRs) in themselves could confer any characteristic C4 expression patterns, 5′- and 3′-UTRs of AhRbcS1 mRNA from the C4 dicot amaranth were linked to a gusA reporter gene. These were constitutively transcribed from a cauliflower mosaic virus promoter and assayed for posttranscriptional expression patterns in transgenic lines of the C4 dicot Flaveria bidentis. Three characteristic C4 expression patterns were conferred by heterologous AhRbcS1 UTRs in transgenic F. bidentis. First, the AhRbcS1 UTRs conferred strong translational enhancement of gusA expression, relative to control constructs lacking these UTRs. Second, while the UTRs did not appear to confer tissue-specific expression when analyzed by β-glucuronidase activity assays, differences in gusA mRNA accumulation were observed in leaves, stems, and roots. Third, the AhRbcS1 UTRs conferred preferential gusA expression (enzyme activity and gusA mRNA accumulation) in leaf bundle sheath cells. AhRbcS1 UTR-mediated translational enhancement was also observed in transgenic C3 plants (tobacco [Nicotiana tabacum]) and in in vitro translation extracts. These mRNAs appear to be translated with different efficiencies in C4 versus C3 plants, indicating that processes determining overall translational efficiency may vary between these two categories of higher plants. Our findings suggest that the AhRbcS1 5′-UTR functions as a strong translational enhancer in leaves and other tissues, and may work synergistically with the 3′-UTR to modulate overall levels of Rubisco gene expression in different tissues and cell types of C4 plants.