JoVE Visualize What is visualize?
Stop Reading. Start Watching.
Advanced Search
Stop Reading. Start Watching.
Regular Search
Find video protocols related to scientific articles indexed in Pubmed.
Phylotranscriptomic analysis of the origin and early diversification of land plants.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 10-29-2014
Show Abstract
Hide Abstract
Reconstructing the origin and evolution of land plants and their algal relatives is a fundamental problem in plant phylogenetics, and is essential for understanding how critical adaptations arose, including the embryo, vascular tissue, seeds, and flowers. Despite advances in molecular systematics, some hypotheses of relationships remain weakly resolved. Inferring deep phylogenies with bouts of rapid diversification can be problematic; however, genome-scale data should significantly increase the number of informative characters for analyses. Recent phylogenomic reconstructions focused on the major divergences of plants have resulted in promising but inconsistent results. One limitation is sparse taxon sampling, likely resulting from the difficulty and cost of data generation. To address this limitation, transcriptome data for 92 streptophyte taxa were generated and analyzed along with 11 published plant genome sequences. Phylogenetic reconstructions were conducted using up to 852 nuclear genes and 1,701,170 aligned sites. Sixty-nine analyses were performed to test the robustness of phylogenetic inferences to permutations of the data matrix or to phylogenetic method, including supermatrix, supertree, and coalescent-based approaches, maximum-likelihood and Bayesian methods, partitioned and unpartitioned analyses, and amino acid versus DNA alignments. Among other results, we find robust support for a sister-group relationship between land plants and one group of streptophyte green algae, the Zygnematophyceae. Strong and robust support for a clade comprising liverworts and mosses is inconsistent with a widely accepted view of early land plant evolution, and suggests that phylogenetic hypotheses used to understand the evolution of fundamental plant traits should be reevaluated.
Related JoVE Video
Multiple Polyploidy Events in the Early Radiation of Nodulating and Nonnodulating Legumes.
Mol. Biol. Evol.
PUBLISHED: 10-27-2014
Show Abstract
Hide Abstract
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.
Related JoVE Video
Pectinmethylesterases (PME) and pectinmethylesterase inhibitors (PMEI) enriched during phloem fiber development in flax (Linum usitatissimum).
PLoS ONE
PUBLISHED: 08-14-2014
Show Abstract
Hide Abstract
Flax phloem fibers achieve their length by intrusive-diffusive growth, which requires them to penetrate the extracellular matrix of adjacent cells. Fiber elongation therefore involves extensive remodelling of cell walls and middle lamellae, including modifying the degree and pattern of methylesterification of galacturonic acid (GalA) residues of pectin. Pectin methylesterases (PME) are important enzymes for fiber elongation as they mediate the demethylesterification of GalA in muro, in either a block-wise fashion or in a random fashion. Our objective was to identify PMEs and PMEIs that mediate phloem fiber elongation in flax. For this purpose, we measured transcript abundance of candidate genes at nine different stages of stem and fiber development and found sets of genes enriched during fiber elongation and maturation as well as during xylem development. We expressed one of the flax PMEIs in E. coli and demonstrated that it was able to inhibit most of the native PME activity in the upper portion of the flax stem. These results identify key genetic components of the intrusive growth process and define targets for fiber engineering and crop improvement.
Related JoVE Video
Horizontal transfer of an adaptive chimeric photoreceptor from bryophytes to ferns.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 04-14-2014
Show Abstract
Hide Abstract
Ferns are well known for their shade-dwelling habits. Their ability to thrive under low-light conditions has been linked to the evolution of a novel chimeric photoreceptor--neochrome--that fuses red-sensing phytochrome and blue-sensing phototropin modules into a single gene, thereby optimizing phototropic responses. Despite being implicated in facilitating the diversification of modern ferns, the origin of neochrome has remained a mystery. We present evidence for neochrome in hornworts (a bryophyte lineage) and demonstrate that ferns acquired neochrome from hornworts via horizontal gene transfer (HGT). Fern neochromes are nested within hornwort neochromes in our large-scale phylogenetic reconstructions of phototropin and phytochrome gene families. Divergence date estimates further support the HGT hypothesis, with fern and hornwort neochromes diverging 179 Mya, long after the split between the two plant lineages (at least 400 Mya). By analyzing the draft genome of the hornwort Anthoceros punctatus, we also discovered a previously unidentified phototropin gene that likely represents the ancestral lineage of the neochrome phototropin module. Thus, a neochrome originating in hornworts was transferred horizontally to ferns, where it may have played a significant role in the diversification of modern ferns.
Related JoVE Video
Identification and functional characterization of a flax UDP-glycosyltransferase glucosylating secoisolariciresinol (SECO) into secoisolariciresinol monoglucoside (SMG) and diglucoside (SDG).
BMC Plant Biol.
PUBLISHED: 03-19-2014
Show Abstract
Hide Abstract
Lignans are a class of diphenolic nonsteroidal phytoestrogens often found glycosylated in planta. Flax seeds are a rich source of secoisolariciresinol diglucoside (SDG) lignans. Glycosylation is a process by which a glycosyl group is covalently attached to an aglycone substrate and is catalyzed by uridine diphosphate glycosyltransferases (UGTs). Until now, very little information was available on UGT genes that may play a role in flax SDG biosynthesis. Here we report on the identification, structural and functional characterization of 5 putative UGTs potentially involved in secoisolariciresinol (SECO) glucosylation in flax.
Related JoVE Video
Identification, expression and interaction analyses of calcium-dependent protein kinase (CPK) genes in canola (Brassica napus L.).
BMC Genomics
PUBLISHED: 03-07-2014
Show Abstract
Hide Abstract
Canola (Brassica napus L.) is one of the most important oil-producing crops in China and worldwide. The yield and quality of canola is frequently threatened by environmental stresses including drought, cold and high salinity. Calcium is a well-known ubiquitous intracellular secondary messenger in plants. Calcium-dependent protein kinases (CPKs) are Ser/Thr protein kinases found only in plants and some protozoans. CPKs are Ca2+ sensors that have both Ca2+ sensing function and kinase activity within a single protein and play crucial roles in plant development and responses to various environmental stresses.
Related JoVE Video
Identification and functional analysis of mitogen-activated protein kinase kinase kinase (MAPKKK) genes in canola (Brassica napus L.).
J. Exp. Bot.
PUBLISHED: 03-06-2014
Show Abstract
Hide Abstract
Mitogen-activated protein kinase (MAPK) signalling cascades, consisting of three types of reversibly phosphorylated kinases (MAPKKK, MAPKK, and MAPK), are involved in important processes including plant immunity and hormone responses. The MAPKKKs comprise the largest family in the MAPK cascades, yet only a few of these genes have been associated with physiological functions, even in the model plant Arabidopsis thaliana. Canola (Brassica napus L.) is one of the most important oilseed crops in China and worldwide. To explore MAPKKK functions in biotic and abiotic stress responses in canola, 66 MAPKKK genes were identified and 28 of them were cloned. Phylogenetic analysis of these canola MAPKKKs with homologous genes from representative species classified them into three groups (A-C), comprising four MAPKKKs, seven ZIKs, and 17 Raf genes. A further 15 interaction pairs between these MAPKKKs and the downstream BnaMKKs were identified through a yeast two-hybrid assay. The interactions were further validated through bimolecular fluorescence complementation (BiFC) analysis. In addition, by quantitative real-time reverse transcription-PCR, it was further observed that some of these BnaMAPKKK genes were regulated by different hormone stimuli, abiotic stresses, or fungal pathogen treatments. Interestingly, two novel BnaMAPKKK genes, BnaMAPKKK18 and BnaMAPKKK19, which could elicit hypersensitive response (HR)-like cell death when transiently expressed in Nicotiana benthamiana leaves, were successfully identified. Moreover, it was found that BnaMAPKKK19 probably mediated cell death through BnaMKK9. Overall, the present work has laid the foundation for further characterization of this important MAPKKK gene family in canola.
Related JoVE Video
Identification and characterization of CBL and CIPK gene families in canola (Brassica napus L.).
BMC Plant Biol.
PUBLISHED: 01-07-2014
Show Abstract
Hide Abstract
Canola (Brassica napus L.) is one of the most important oil-producing crops in China and worldwide. The yield and quality of canola is frequently threatened by environmental stresses including drought, cold and high salinity. Calcium is a ubiquitous intracellular secondary messenger in plants. Calcineurin B-like proteins (CBLs) are Ca2+ sensors and regulate a group of Ser/Thr protein kinases called CBL-interacting protein kinases (CIPKs). Although the CBL-CIPK network has been demonstrated to play crucial roles in plant development and responses to various environmental stresses in Arabidopsis, little is known about their function in canola.
Related JoVE Video
Chitinase-like (CTL) and cellulose synthase (CESA) gene expression in gelatinous-type cellulosic walls of flax (Linum usitatissimum L.) bast fibers.
PLoS ONE
PUBLISHED: 01-01-2014
Show Abstract
Hide Abstract
Plant chitinases (EC 3.2.1.14) and chitinase-like (CTL) proteins have diverse functions including cell wall biosynthesis and disease resistance. We analyzed the expression of 34 chitinase and chitinase-like genes of flax (collectively referred to as LusCTLs), belonging to glycoside hydrolase family 19 (GH19). Analysis of the transcript expression patterns of LusCTLs in the stem and other tissues identified three transcripts (LusCTL19, LusCTL20, LusCTL21) that were highly enriched in developing bast fibers, which form cellulose-rich gelatinous-type cell walls. The same three genes had low relative expression in tissues with primary cell walls and in xylem, which forms a xylan type of secondary cell wall. Phylogenetic analysis of the LusCTLs identified a flax-specific sub-group that was not represented in any of other genomes queried. To provide further context for the gene expression analysis, we also conducted phylogenetic and expression analysis of the cellulose synthase (CESA) family genes of flax, and found that expression of secondary wall-type LusCESAs (LusCESA4, LusCESA7 and LusCESA8) was correlated with the expression of two LusCTLs (LusCTL1, LusCTL2) that were the most highly enriched in xylem. The expression of LusCTL19, LusCTL20, and LusCTL21 was not correlated with that of any CESA subgroup. These results defined a distinct type of CTLs that may have novel functions specific to the development of the gelatinous (G-type) cellulosic walls.
Related JoVE Video
Differential expression of miRNAs in Brassica napus root following infection with Plasmodiophora brassicae.
PLoS ONE
PUBLISHED: 01-01-2014
Show Abstract
Hide Abstract
Canola (oilseed rape, Brassica napus L.) is susceptible to infection by the biotrophic protist Plasmodiophora brassicae, the causal agent of clubroot. To understand the roles of microRNAs (miRNAs) during the post-transcriptional regulation of disease initiation and progression, we have characterized the changes in miRNA expression profiles in canola roots during clubroot disease development and have compared these to uninfected roots. Two different stages of clubroot development were targeted in this miRNA profiling study: an early time of 10-dpi for disease initiation and a later 20-dpi, by which time the pathogen had colonized the roots (as evident by visible gall formation and histological observations). P. brassicae responsive miRNAs were identified and validated by qRT-PCR of miRNAs and the subsequent validation of the target mRNAs through starBase degradome analysis, and through 5' RLM-RACE. This study identifies putative miRNA-regulated genes with roles during clubroot disease initiation and development. Putative target genes identified in this study included: transcription factors (TFs), hormone-related genes, as well as genes associated with plant stress response regulation such as cytokinin, auxin/ethylene response elements. The results of our study may assist in elucidating the role of miRNAs in post-transcriptional regulation of target genes during disease development and may contribute to the development of strategies to engineer durable resistance to this important phytopathogen.
Related JoVE Video
Phylogenetic pinpointing of a paleopolyploidy event within the flax genus (Linum) using transcriptomics.
Ann. Bot.
PUBLISHED: 12-30-2013
Show Abstract
Hide Abstract
Background and AimsCultivated flax (Linum usitatissimum) is known to have undergone a whole-genome duplication around 5-9 million years ago. The aim of this study was to investigate whether other whole-genome duplication events have occurred in the evolutionary history of cultivated flax. Knowledge of such whole-genome duplications will be important in understanding the biology and genomics of cultivated flax.MethodsTranscriptomes of 11 Linum species were sequenced using the Illumina platform. The short reads were assembled de novo and the DupPipe pipeline was used to look for signatures of polyploidy events from the age distribution of paralogues. In addition, phylogenies of all paralogues were assembled within an estimated age window of interest. These phylogenies were assessed for evidence of a paleopolyploidy event within the genus Linum.Key ResultsA previously unknown paleopolyploidy event that occurred 20-40 million years ago was discovered and shown to be specific to a clade within Linum containing cultivated flax (L. usitatissimum) and other mainly blue-flowered species. The finding was supported by two lines of evidence. First, a significant change of slope (peak) was shown in the age distribution of paralogues that was phylogenetically restricted to, and ubiquitous in, this clade. Second, a large number of paralogue phylogenies were retrieved that are consistent with a polyploidy event occurring within that clade.ConclusionsThe results show the utility of multi-species transcriptomics for detecting whole-genome duplication events and demonstrate that that multiple rounds of polyploidy have been important in shaping the evolutionary history of flax. Understanding and characterizing these whole-genome duplication events will be important for future Linum research.
Related JoVE Video
Characterization and transcript profiling of the pectin methylesterase (PME) and pectin methylesterase inhibitor (PMEI) gene families in flax (Linum usitatissimum).
BMC Genomics
PUBLISHED: 08-03-2013
Show Abstract
Hide Abstract
Pectin methylesterases (PMEs) catalyze the demethylesterification of homogalacturonans in the cell wall; their activity is regulated in part by pectin methylesterase inhibitors (PMEIs). PME activity may result in either rigidification or loosening of the cell wall, depending on the mode of demethylesterification. The activity of PMEs in the middle lamella is expected to affect intrusive elongation of phloem fibers, and their adhesion to adjacent cells. Length and extractability of phloem fibers are qualities important for their industrial uses in textiles and composites. As only three flax PMEs had been previously described, we were motivated to characterize the PME and PMEI gene families of flax.
Related JoVE Video
Identification and analysis of MKK and MPK gene families in canola (Brassica napus L.).
BMC Genomics
PUBLISHED: 02-22-2013
Show Abstract
Hide Abstract
Eukaryotic mitogen-activated protein kinase (MAPK/MPK) signaling cascades transduce and amplify environmental signals via three types of reversibly phosphorylated kinases to activate defense gene expression. Canola (oilseed rape, Brassica napus) is a major crop in temperate regions. Identification and characterization of MAPK and MAPK kinases (MAPKK/MKK) of canola will help to elucidate their role in responses to abiotic and biotic stresses.
Related JoVE Video
Genomic and expression analysis of the flax (Linum usitatissimum) family of glycosyl hydrolase 35 genes.
BMC Genomics
PUBLISHED: 01-17-2013
Show Abstract
Hide Abstract
Several ?-galactosidases of the Glycosyl Hydrolase 35 (GH35) family have been characterized, and many of these modify cell wall components, including pectins, xyloglucans, and arabinogalactan proteins. The phloem fibres of flax (Linum usitatissimum) have gelatinous-type cell walls that are rich in crystalline cellulose and depend on ?-galactosidase activity for their normal development. In this study, we investigate the transcript expression patterns and inferred evolutionary relationships of the complete set of flax GH35 genes, to better understand the functions of these genes in flax and other species.
Related JoVE Video
Ecological implications of single and mixed nitrogen nutrition in Arabidopsis thaliana.
BMC Ecol.
PUBLISHED: 01-10-2013
Show Abstract
Hide Abstract
Ecologists recognize that plants capture nitrogen in many chemical forms that include amino acids. Access to multiple nitrogen types in plant communities has been argued to enhance plant performance, access to nitrogen and alter ecological interactions in ways that may promote species coexistence. However, data supporting these arguments have been limited. While it is known that plants uptake amino acids from soil, long term studies that link amino acid uptake to measures of plant performance and potential reproductive effort are not typically performed. Here, a series of experiments that link uptake of nitrate, glutamine or asparagine with lifetime reproductive effort in Arabidopsis thaliana are reported. Nitrogen was offered either singly or in mixture and at a variety of combinations. Traits related to reproductive output were measured, as was the preference for each type of nitrogen.
Related JoVE Video
LuFLA1PRO and LuBGAL1PRO promote gene expression in the phloem fibres of flax (Linum usitatissimum).
Plant Cell Rep.
PUBLISHED: 01-02-2013
Show Abstract
Hide Abstract
Cell type-specific promoters were identified that drive gene expression in an industrially important product. To identify flax (Linum usitatissimum) gene promoters, we analyzed the genomic regions upstream of a fasciclin-like arabinogalactan protein (LuFLA1) and a beta-galactosidase (LuBGAL1). Both of these genes encode transcripts that have been found to be highly enriched in tissues bearing phloem fibres. Using a beta-glucuronidase (GUS) reporter construct, we found that a 908-bp genomic sequence upstream of LuFLA1 (LuFLA1PRO) directed GUS expression with high specificity to phloem fibres undergoing secondary cell wall development. The DNA sequence upstream of LuBGAL1 (LuBGAL1PRO) likewise produced GUS staining in phloem fibres with developing secondary walls, as well as in tissues of developing flowers and seed bolls. These data provide further evidence of a specific role for LuFLA1 in phloem fibre development, and demonstrate the utility of LuFLA1PRO and LuBGAL1PRO as tools for biotechnology and further investigations of phloem fibre development.
Related JoVE Video
Development of cellulosic secondary walls in flax fibers requires beta-galactosidase.
Plant Physiol.
PUBLISHED: 05-19-2011
Show Abstract
Hide Abstract
Bast (phloem) fibers, tension wood fibers, and other cells with gelatinous-type secondary walls are rich in crystalline cellulose. In developing bast fibers of flax (Linum usitatissimum), a galactan-enriched matrix (Gn-layer) is gradually modified into a mature cellulosic gelatinous-layer (G-layer), which ultimately comprises most of the secondary cell wall. Previous studies have correlated this maturation process with expression of a putative ?-galactosidase. Here, we demonstrate that ?-galactosidase activity is in fact necessary for the dynamic remodeling of polysaccharides that occurs during normal secondary wall development in flax fibers. We found that developing stems of transgenic (LuBGAL-RNAi) flax with reduced ?-galactosidase activity had lower concentrations of free Gal and had significant reductions in the thickness of mature cellulosic G-layers compared with controls. Conversely, Gn-layers, labeled intensively by the galactan-specific LM5 antibody, were greatly expanded in LuBGAL-RNAi transgenic plants. Gross morphology and stem anatomy, including the thickness of bast fiber walls, were otherwise unaffected by silencing of ?-galactosidase transcripts. These results demonstrate a specific requirement for ?-galactosidase in hydrolysis of galactans during formation of cellulosic G-layers. Transgenic lines with reduced ?-galactosidase activity also had biochemical and spectroscopic properties consistent with a reduction in cellulose crystallinity. We further demonstrated that the tensile strength of normal flax stems is dependent on ?-galactosidase-mediated development of the phloem fiber G-layer. Thus, the mechanical strength that typifies flax stems is dependent on a thick, cellulosic G-layer, which itself depends on ?-galactosidase activity within the precursor Gn-layer. These observations demonstrate a novel role for matrix polysaccharides in cellulose deposition; the relevance of these observations to the development of cell walls in other species is also discussed.
Related JoVE Video
PELPK1 (At5g09530) contains a unique pentapeptide repeat and is a positive regulator of germination in Arabidopsis thaliana.
Plant Cell Rep.
PUBLISHED: 03-19-2011
Show Abstract
Hide Abstract
Arabidopsis gene At5g09530 has been previously annotated as a cell wall protein of either the hydroxyproline-rich glycoprotein (HRGP), extensin-like, or proline-rich protein families (e.g. PRP10). However, At5g09530 shows important differences between its amino acid sequence and these other proteins. At5g09530 lacks any motifs typical of major groups of cell wall proteins, but contains 36 repeats of a unique pentapeptide (Pro-Glu-Leu|Ile|Val-Pro-Lys), which we have named the PELPK motif. This motif is repeated in only one other Arabidopsis protein (At5g09520), but proteins containing repeated PELPK motifs are found in many other angiosperms. At5g09530 is predicted to encode an intrinsically disordered protein. We characterized the phenotype of transgenic Arabidopsis with either reduced (RNAi) or increased constitutive (35S promoter) transcript expression of At5g09530. RNAi lines exhibited significantly slower germination and root growth, while overexpression lines had accelerated germination and root growth compared to wild type. Similarly, when grown on soil, RNAi lines had delayed growth and flowering, while overexpression lines had accelerated growth and flowering as compared to wild type. Based on amino acid composition, the presence of a distinct repeated pentapeptide motif and predicted intrinsically disordered structure, we conclude that At5g09530 is not an HRGP, PRP, or extensin-like protein. Because At5g09530 is a distinct and conserved protein, we propose to name it PELPK1, and to name its presumptive inparalog (At5g09520) PELPK2. PELPK1 is necessary for normal rates of germination and growth, while overexpression of PELPK1 is sufficient to accelerate germination and growth.
Related JoVE Video
Gene expression analysis of flax seed development.
BMC Plant Biol.
PUBLISHED: 02-21-2011
Show Abstract
Hide Abstract
Flax, Linum usitatissimum L., is an important crop whose seed oil and stem fiber have multiple industrial applications. Flax seeds are also well-known for their nutritional attributes, viz., omega-3 fatty acids in the oil and lignans and mucilage from the seed coat. In spite of the importance of this crop, there are few molecular resources that can be utilized toward improving seed traits. Here, we describe flax embryo and seed development and generation of comprehensive genomic resources for the flax seed.
Related JoVE Video
Exploration of inhibitors for diaminopimelate aminotransferase.
Bioorg. Med. Chem.
PUBLISHED: 01-31-2010
Show Abstract
Hide Abstract
Bacteria and higher plants make l-lysine from diaminopimelic acid (DAP). In mammals l-lysine is an essential amino acid that must be acquired from the diet as the biosynthetic pathway is absent for this key constituent of proteins. Recently, ll-diaminopimelate aminotransferase (ll-DAP-AT), a pyridoxal-5-phosphate (PLP)-dependent enzyme, was reported to catalyze a key step in the route to l-lysine in plants and Chlamydia. Specific inhibitors of this enzyme could thus potentially serve as herbicides or antibiotics that are non-toxic to mammals. In this work, 29,201 inhibitors were screened against ll-DAP-AT and the IC(50) values were determined for the top 46 compounds. An aryl hydrazide and rhodanine derivatives were further modified to generate 20 analogues that were also tested against ll-DAP-AT. These analogues provide additional structure-activity relationships (SAR) that are useful in guiding further design of inhibitors.
Related JoVE Video
Transcriptome analysis of secondary-wall-enriched seed coat tissues of canola (Brassica napus L.).
Plant Cell Rep.
PUBLISHED: 01-18-2010
Show Abstract
Hide Abstract
The seed coat of Brassica napus (canola, oilseed rape) is derived from ovule integuments and contains a layer of palisade cells, which have thick secondary walls. Because cellulosic walls and other indigestible components of the seed coat contribute negatively to the value of oilseeds, efforts are underway to alter seed development. To facilitate these efforts, and to better understand the biology of seed coats, we used a 90,000 element microarray to identify genes whose transcripts were expressed in developing seed coats of B. napus. After dissecting seed coats into three layers, and comparing transcript expression in the middle fraction (which contained the palisade-enriched tissue and bulk of inner integument) to transcript expression in developing hypocotyls, we identified 674 genes whose transcripts were more abundant in the middle fraction of the seed coat. Among these were well-characterized markers of seed coat identity and many genes associated with metabolism of cell wall polysaccharides, flavonoids and various cell wall proteins and transcription factors. Conversely, we identified 1,203 genes whose transcripts were more abundant in the hypocotyl tissue as compared to seed coat, including xylem-specific markers, such as XCP1 and XCP2. We validated 21 of the differentially expressed transcripts using quantitative RT-PCR. The results define a set of transcripts that are highly enriched in the developing seed coat of B. napus.
Related JoVE Video
Making the most of drought and salinity transcriptomics.
Plant Cell Environ.
PUBLISHED: 11-25-2009
Show Abstract
Hide Abstract
More than 100 different studies of plant transcriptomic responses to salinity or drought-related stress have now been published. Most of these use microarrays or related high-throughput profiling technologies. This compels us to ask three questions in review: (1) what has transcriptomics contributed to our understanding of stress physiology; (2) what limits the ability of transcriptomics to contribute to increases in stress tolerance; and (3) given these limits, what are the most appropriate uses of transcriptomics? We conclude that although microarrays are now a mature technology that accurately describes the transcriptome, the consistently low correlation between transcript abundance and other measures of gene expression imposes an inherent limitation that cannot be ignored. Further limitations on the relevance of transcriptomics arise in some cases from experimental practices related to the treatment regimen and the selection of tissue or germplasm. Nevertheless, there is good evidence to support the continued use of transcriptomics, especially emerging techniques such as RNA-Seq, as a screening tool for candidate gene discovery. Microarrays can also be valuable in analysing the transcriptome per se (e.g. when describing the phenotype of a transcription factor mutant or discovering non-coding RNA species), and when integrated with other types of data including metabolomic analyses.
Related JoVE Video
Identification and expression analysis of WRKY transcription factor genes in canola (Brassica napus L.) in response to fungal pathogens and hormone treatments.
BMC Plant Biol.
PUBLISHED: 06-03-2009
Show Abstract
Hide Abstract
Members of plant WRKY transcription factor families are widely implicated in defense responses and various other physiological processes. For canola (Brassica napus L.), no WRKY genes have been described in detail. Because of the economic importance of this crop, and its evolutionary relationship to Arabidopsis thaliana, we sought to characterize a subset of canola WRKY genes in the context of pathogen and hormone responses.
Related JoVE Video
Functional characterization of Arabidopsis NaCl-inducible WRKY25 and WRKY33 transcription factors in abiotic stresses.
Plant Mol. Biol.
PUBLISHED: 04-15-2009
Show Abstract
Hide Abstract
Previous microarray analyses of Arabidopsis roots identified two closely related WRKY transcription factors (WRKY25 and WRKY33) among the transcripts that increased in abundance following treatment with NaCl. Here, we report further characterization of these genes, which we found to be inducible by a variety of abiotic stresses in an SOS-pathway independent manner, although WRKY33 induction was dependent on ABA signaling. Transcripts of both genes were detected in roots and leaves, while specific patterns of enrichment were observed in stems and floral buds for WRKY25 and WRKY33, respectively. We also identified upstream intergenic regions from each gene that were sufficient to confer stress-inducible expression on a reporter gene. However, the stress sensitivity of wrky25 null mutants did not differ from wild-type under any assay condition, while wrky33 null mutants and wrky25wrky33 double mutants showed only a moderate increase in NaCl-sensitivity, suggesting functional redundancy with other transcription factors. Nevertheless, overexpression of WRKY25 or WRKY33 was sufficient to increase Arabidopsis NaCl tolerance, while increasing sensitivity to ABA. Through microarray analyses of relevant genotypes, we identified 31 and 208 potential downstream targets of WRKY25 and WRKY33, respectively, most of which contained a W-box in their upstream regions.
Related JoVE Video
Functional characterization of the Arabidopsis bHLH92 transcription factor in abiotic stress.
Mol. Genet. Genomics
PUBLISHED: 01-25-2009
Show Abstract
Hide Abstract
In our previous microarray analysis of NaCl-treated Arabidopsis roots, we identified a basic-helix-loop-helix (bHLH) transcription factor, bHLH92 (At5g43650), as one of the transcripts showing the greatest fold-increase in abundance upon NaCl exposure. Here, we characterize the role of bHLH92 in the context of abiotic stress physiology and hormone responses. We observed that bHLH92 transcript abundance increases in response to NaCl, dehydration, mannitol, and cold treatments, and compared these responses to those of two closely related genes: bHLH41 and bHLH42. The NaCl-inducibility of bHLH92 was only partially dependent on abscisic acid (ABA) biosynthesis and SALT OVERLY SENSITIVE2 (SOS2) pathways. As compared to WT, root elongation of bhlh92 mutants was more sensitive to mannitol, and these mutants also showed increased electrolyte leakage following NaCl treatments. Overexpression of bHLH92 moderately increased the tolerance to NaCl and osmotic stresses. Finally, we identified at least 19 putative downstream target genes of bHLH92 under NaCl treatment using an oligonucleotide microarray. Together these data show that bHLH92 functions in plant responses to osmotic stresses, although the net contribution of bHLH92-regulated genes to stress tolerance appears relatively limited in proportion to what might be expected from its transcript expression pattern.
Related JoVE Video
Evaluating methods for isolating total RNA and predicting the success of sequencing phylogenetically diverse plant transcriptomes.
PLoS ONE
Show Abstract
Hide Abstract
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.
Related JoVE Video
Identification, characterization and distribution of transposable elements in the flax (Linum usitatissimum L.) genome.
BMC Genomics
Show Abstract
Hide Abstract
Flax (Linum usitatissimum L.) is an important crop for the production of bioproducts derived from its seed and stem fiber. Transposable elements (TEs) are widespread in plant genomes and are a key component of their evolution. The availability of a genome assembly of flax (Linum usitatissimum) affords new opportunities to explore the diversity of TEs and their relationship to genes and gene expression.
Related JoVE Video
Over-expression of a novel JAZ family gene from Glycine soja, increases salt and alkali stress tolerance.
Biochem. Biophys. Res. Commun.
Show Abstract
Hide Abstract
Salt and alkali stress are two of the main environmental factors limiting crop production. Recent discoveries show that the JAZ family encodes plant-specific genes involved in jasmonate signaling. However, there is only limited information about this gene family in abiotic stress response, and in wild soybean (Glycine soja), which is a species noted for its tolerance to alkali and salinity. Here, we isolated and characterized a novel JAZ family gene, GsJAZ2, from G. soja. Transcript abundance of GsJAZ2 increased following exposure to salt, alkali, cold and drought. Over-expression of GsJAZ2 in Arabidopsis resulted in enhanced plant tolerance to salt and alkali stress. The expression levels of some alkali stress response and stress-inducible marker genes were significantly higher in the GsJAZ2 overexpression lines as compared to wild-type plants. Subcellular localization studies using a GFP fusion protein showed that GsJAZ2 was localized to the nucleus. These results suggest that the newly isolated wild soybean GsJAZ2 is a positive regulator of plant salt and alkali stress tolerance.
Related JoVE Video
Modeling the yew tree tubulin and a comparison of its interaction with paclitaxel to human tubulin.
Pharm. Res.
Show Abstract
Hide Abstract
To explore possible ways in which yew tree tubulin is naturally resistant to paclitaxel. While the yew produces a potent cytotoxin, paclitaxel, it is immune to paclitaxels cytotoxic action.
Related JoVE Video
The genome of flax (Linum usitatissimum) assembled de novo from short shotgun sequence reads.
Plant J.
Show Abstract
Hide Abstract
Flax (Linum usitatissimum) is an ancient crop that is widely cultivated as a source of fiber, oil and medicinally relevant compounds. To accelerate crop improvement, we performed whole-genome shotgun sequencing of the nuclear genome of flax. Seven paired-end libraries ranging in size from 300 bp to 10 kb were sequenced using an Illumina genome analyzer. A de novo assembly, comprised exclusively of deep-coverage (approximately 94× raw, approximately 69× filtered) short-sequence reads (44-100 bp), produced a set of scaffolds with N(50) =694 kb, including contigs with N(50)=20.1 kb. The contig assembly contained 302 Mb of non-redundant sequence representing an estimated 81% genome coverage. Up to 96% of published flax ESTs aligned to the whole-genome shotgun scaffolds. However, comparisons with independently sequenced BACs and fosmids showed some mis-assembly of regions at the genome scale. A total of 43384 protein-coding genes were predicted in the whole-genome shotgun assembly, and up to 93% of published flax ESTs, and 86% of A. thaliana genes aligned to these predicted genes, indicating excellent coverage and accuracy at the gene level. Analysis of the synonymous substitution rates (K(s) ) observed within duplicate gene pairs was consistent with a recent (5-9 MYA) whole-genome duplication in flax. Within the predicted proteome, we observed enrichment of many conserved domains (Pfam-A) that may contribute to the unique properties of this crop, including agglutinin proteins. Together these results show that de novo assembly, based solely on whole-genome shotgun short-sequence reads, is an efficient means of obtaining nearly complete genome sequence information for some plant species.
Related JoVE Video

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.