Showing papers in "BMC Plant Biology in 2009"

Nanoparticle penetration and transport in living pumpkin plants: in situ subcellular identification

Abstract: In recent years, the application of nanotechnology in several fields of bioscience and biomedicine has been studied. The use of nanoparticles for the targeted delivery of substances has been given special attention and is of particular interest in the treatment of plant diseases. In this work both the penetration and the movement of iron-carbon nanoparticles in plant cells have been analyzed in living plants of Cucurbita pepo. The nanoparticles were applied in planta using two different application methods, injection and spraying, and magnets were used to retain the particles in movement in specific areas of the plant. The main experimental approach, using correlative light and electron microscopy provided evidence of intracellular localization of nanoparticles and their displacement from the application point. Long range movement of the particles through the plant body was also detected, particles having been found near the magnets used to immobilize and concentrate them. Furthermore, cell response to the nanoparticle presence was detected. Nanoparticles were capable of penetrating living plant tissues and migrating to different regions of the plant, although movements over short distances seemed to be favoured. These findings show that the use of carbon coated magnetic particles for directed delivery of substances into plant cells is a feasible application.

A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat.

Abstract: Wheat (Triticum ssp.) is an important food source for humans in many regions around the world. However, the ability to understand and modify gene function for crop improvement is hindered by the lack of available genomic resources. TILLING is a powerful reverse genetics approach that combines chemical mutagenesis with a high-throughput screen for mutations. Wheat is specially well-suited for TILLING due to the high mutation densities tolerated by polyploids, which allow for very efficient screens. Despite this, few TILLING populations are currently available. In addition, current TILLING screening protocols require high-throughput genotyping platforms, limiting their use. We developed mutant populations of pasta and common wheat and organized them for TILLING. To simplify and decrease costs, we developed a non-denaturing polyacrylamide gel set-up that uses ethidium bromide to detect fragments generated by crude celery juice extract digestion of heteroduplexes. This detection method had similar sensitivity as traditional LI-COR screens, suggesting that it represents a valid alternative. We developed genome-specific primers to circumvent the presence of multiple homoeologous copies of our target genes. Each mutant library was characterized by TILLING multiple genes, revealing high mutation densities in both the hexaploid (~1/38 kb) and tetraploid (~1/51 kb) populations for 50% GC targets. These mutation frequencies predict that screening 1,536 lines for an effective target region of 1.3 kb with 50% GC content will result in ~52 hexaploid and ~39 tetraploid mutant alleles. This implies a high probability of obtaining knock-out alleles (P = 0.91 for hexaploid, P = 0.84 for tetraploid), in addition to multiple missense mutations. In total, we identified over 275 novel alleles in eleven targeted gene/genome combinations in hexaploid and tetraploid wheat and have validated the presence of a subset of them in our seed stock. We have generated reverse genetics TILLING resources for pasta and bread wheat and achieved a high mutation density in both populations. We also developed a modified screening method that will lower barriers to adopt this promising technology. We hope that the use of this reverse genetics resource will enable more researchers to pursue wheat functional genomics and provide novel allelic diversity for wheat improvement.

Medicago truncatula and Glomus intraradices gene expression in cortical cells harboring arbuscules in the arbuscular mycorrhizal symbiosis

Abstract: Most vascular flowering plants have the capacity to form symbiotic associations with arbuscular mycorrhizal (AM) fungi. The symbiosis develops in the roots where AM fungi colonize the root cortex and form arbuscules within the cortical cells. Arbuscules are enveloped in a novel plant membrane and their establishment requires the coordinated cellular activities of both symbiotic partners. The arbuscule-cortical cell interface is the primary functional interface of the symbiosis and is of central importance in nutrient exchange. To determine the molecular events the underlie arbuscule development and function, it is first necessary to identify genes that may play a role in this process. Toward this goal we used the Affymetrix GeneChip® Medicago Genome Array to document the M. truncatula transcript profiles associated with AM symbiosis, and then developed laser microdissection (LM) of M. truncatula root cortical cells to enable analyses of gene expression in individual cell types by RT-PCR. This approach led to the identification of novel M. truncatula and G. intraradices genes expressed in colonized cortical cells and in arbuscules. Within the arbuscule, expression of genes associated with the urea cycle, amino acid biosynthesis and cellular autophagy was detected. Analysis of gene expression in the colonized cortical cell revealed up-regulation of a lysine motif (LysM)-receptor like kinase, members of the GRAS transcription factor family and a symbiosis-specific ammonium transporter that is a likely candidate for mediating ammonium transport in the AM symbiosis. Transcript profiling using the Affymetrix GeneChip® Medicago Genome Array provided new insights into gene expression in M. truncatula roots during AM symbiosis and revealed the existence of several G. intraradices genes on the M. truncatula GeneChip®. A laser microdissection protocol that incorporates low-melting temperature Steedman's wax, was developed to enable laser microdissection of M. truncatula root cortical cells. LM coupled with RT-PCR provided spatial gene expression information for both symbionts and expanded current information available for gene expression in cortical cells containing arbuscules.

Transcript and proteomic analysis of developing white lupin (Lupinus albus L.) roots

Abstract: White lupin (Lupinus albus L.) roots efficiently take up and accumulate (heavy) metals, adapt to phosphate deficiency by forming cluster roots, and secrete antimicrobial prenylated isoflavones during development. Genomic and proteomic approaches were applied to identify candidate genes and proteins involved in antimicrobial defense and (heavy) metal uptake and translocation. A cDNA library was constructed from roots of white lupin seedlings. Eight thousand clones were randomly sequenced and assembled into 2,455 unigenes, which were annotated based on homologous matches in the NCBInr protein database. A reference map of developing white lupin root proteins was established through 2-D gel electrophoresis and peptide mass fingerprinting. High quality peptide mass spectra were obtained for 170 proteins. Microsomal membrane proteins were separated by 1-D gel electrophoresis and identified by LC-MS/MS. A total of 74 proteins were putatively identified by the peptide mass fingerprinting and the LC-MS/MS methods. Genomic and proteomic analyses identified candidate genes and proteins encoding metal binding and/or transport proteins, transcription factors, ABC transporters and phenylpropanoid biosynthetic enzymes. The combined EST and protein datasets will facilitate the understanding of white lupin's response to biotic and abiotic stresses and its utility for phytoremediation. The root ESTs provided 82 perfect simple sequence repeat (SSR) markers with potential utility in breeding white lupin for enhanced agronomic traits.

Differential patterns of reactive oxygen species and antioxidative mechanisms during atrazine injury and sucrose-induced tolerance in Arabidopsis thaliana plantlets

Abstract: Besides being essential for plant structure and metabolism, soluble carbohydrates play important roles in stress responses. Sucrose has been shown to confer to Arabidopsis seedlings a high level of tolerance to the herbicide atrazine, which causes reactive oxygen species (ROS) production and oxidative stress. The effects of atrazine and of exogenous sucrose on ROS patterns and ROS-scavenging systems were studied. Simultaneous analysis of ROS contents, expression of ROS-related genes and activities of ROS-scavenging enzymes gave an integrative view of physiological state and detoxifying potential under conditions of sensitivity or tolerance. Toxicity of atrazine could be related to inefficient activation of singlet oxygen (1O2) quenching pathways leading to 1O2 accumulation. Atrazine treatment also increased hydrogen peroxide (H2O2) content, while reducing gene expressions and enzymatic activities related to two major H2O2-detoxification pathways. Conversely, sucrose-protected plantlets in the presence of atrazine exhibited efficient 1O2 quenching, low 1O2 accumulation and active H2O2-detoxifying systems. In conclusion, sucrose protection was in part due to activation of specific ROS scavenging systems with consequent reduction of oxidative damages. Importance of ROS combination and potential interferences of sucrose, xenobiotic and ROS signalling pathways are discussed.

Genome-wide analysis of major intrinsic proteins in the tree plant Populus trichocarpa : Characterization of XIP subfamily of aquaporins from evolutionary perspective

Abstract: Members of major intrinsic proteins (MIPs) include water-conducting aquaporins and glycerol-transporting aquaglyceroporins MIPs play important role in plant-water relations The model plants Arabidopsis thaliana, rice and maize contain more than 30 MIPs and based on phylogenetic analysis they can be divided into at least four subfamilies Populus trichocarpa is a model tree species and provides an opportunity to investigate several tree-specific traits In this study, we have investigated Populus MIPs (PtMIPs) and compared them with their counterparts in Arabidopsis, rice and maize Fifty five full-length MIPs have been identified in Populus genome Phylogenetic analysis reveals that Populus has a fifth uncharacterized subfamily (XIPs) Three-dimensional models of all 55 PtMIPs were constructed using homology modeling technique Aromatic/arginine (ar/R) selectivity filters, characteristics of loops responsible for solute selectivity (loop C) and gating (loop D) and group conservation of small and weakly polar interfacial residues have been analyzed Majority of the non-XIP PtMIPs are similar to those in Arabidopsis, rice and maize Additional XIPs were identified from database search and 35 XIP sequences from dicots, fungi, moss and protozoa were analyzed Ar/R selectivity filters of dicots XIPs are more hydrophobic compared to fungi and moss XIPs and hence they are likely to transport hydrophobic solutes Loop C is longer in one of the subgroups of dicot XIPs and most probably has a significant role in solute selectivity Loop D in dicot XIPs has higher number of basic residues Intron loss is observed on two occasions: once between two subfamilies of eudicots and monocot and in the second instance, when dicot and moss XIPs diverged from fungi Expression analysis of Populus MIPs indicates that Populus XIPs don't show any tissue-specific transcript abundance Due to whole genome duplication, Populus has the largest number of MIPs identified in any single species Non-XIP MIPs are similar in all four plant species considered in this study Small and weakly polar residues at the helix-helix interface are group conserved presumably to maintain the hourglass fold of MIP channels Substitutions in ar/R selectivity filter, insertion/deletion in loop C, increasing basic nature of loop D and loss of introns are some of the events occurred during the evolution of dicot XIPs

Comparison of the transcriptomes of American chestnut (Castanea dentata) and Chinese chestnut (Castanea mollissima) in response to the chestnut blight infection.

Abstract: American chestnut (Castanea dentata) was devastated by an exotic pathogen in the beginning of the twentieth century. This chestnut blight is caused by Cryphonectria parasitica, a fungus that infects stem tissues and kills the trees by girdling them. Because of the great economic and ecological value of this species, significant efforts have been made over the century to combat this disease, but it wasn't until recently that a focused genomics approach was initiated. Prior to the Genomic Tool Development for the Fagaceae project, genomic resources available in public databases for this species were limited to a few hundred ESTs. To identify genes involved in resistance to C. parasitica, we have sequenced the transcriptome from fungal infected and healthy stem tissues collected from blight-sensitive American chestnut and blight-resistant Chinese chestnut (Castanea mollissima) trees using ultra high throughput pyrosequencing. We produced over a million 454 reads, totaling over 250 million bp, from which we generated 40,039 and 28,890 unigenes in total from C. mollissima and C. dentata respectively. The functions of the unigenes, from GO annotation, cover a diverse set of molecular functions and biological processes, among which we identified a large number of genes associated with resistance to stresses and response to biotic stimuli. In silico expression analyses showed that many of the stress response unigenes were expressed more in canker tissues versus healthy stem tissues in both American and Chinese chestnut. Comparative analysis also identified genes belonging to different pathways of plant defense against biotic stresses that are differentially expressed in either American or Chinese chestnut canker tissues. Our study resulted in the identification of a large set of cDNA unigenes from American chestnut and Chinese chestnut. The ESTs and unigenes from this study constitute an important resource to the scientific community interested in the discovery of genes involved in various biological processes in Chestnut and other species. The identification of many defense-related genes differentially expressed in canker vs. healthy stem in chestnuts provides many new candidate genes for developing resistance to the chestnut blight and for studying pathways involved in responses of trees to necrotrophic pathogens. We also identified several candidate genes that may underline the difference in resistance to Cryphonectria parasitica between American chestnut and Chinese chestnut.

Over-expression of miR172 causes loss of spikelet determinacy and floral organ abnormalities in rice (Oryza sativa)

Abstract: Regulation of gene expression by microRNAs (miRNAs) plays a crucial role in many developmental and physiological processes in plants. miRNAs act to repress expression of their target genes via mRNA cleavage or translational repression. Dozens of miRNA families have been identified in rice, 21 of which are conserved between rice and Arabidopsis. miR172 is a conserved miRNA family which has been shown to regulate expression of APETALA2 (AP2)-like transcription factors in Arabidopsis and maize. The rice genome encodes five AP2-like genes predicted to be targets of miR172. To determine whether these rice AP2-like genes are regulated by miR172 and investigate the function of the target genes, we studied the effect of over-expressing two members of the miR172 family on rice plant development. Analysis of miR172 expression showed that it is most highly expressed in late vegetative stages and developing panicles. Analyses of expression of three miR172 targets showed that SUPERNUMERARY BRACT (SNB) and Os03g60430 have high expression in developing panicles. Expression of miR172 was not inversely correlated with expression of its targets although miR172-mediated cleavage of SNB was detected by 5' rapid amplification of cDNA ends (RACE). Over-expression of miR172b in rice delayed the transition from spikelet meristem to floral meristem, and resulted in floral and seed developmental defects, including changes to the number and identity of floral organs, lower fertility and reduced seed weight. Plants over-expressing miR172b not only phenocopied the T-DNA insertion mutant of SNB but showed additional defects in floret development not seen in the snb mutant. However SNB expression was not reduced in the miR172b over-expression plants. The phenotypes resulting from over-expression of miR172b suggests it represses SNB and at least one of the other miR172 targets, most likely Os03g60430, indicating roles for other AP2-like genes in rice floret development. miR172 and the AP2-like genes had overlapping expression patterns in rice and their expression did not show an obvious negative correlation. There was not a uniform decrease in the expression of the AP2-like miR172 target mRNAs in the miR172b over-expression plants. These observations are consistent with miR172 functioning via translational repression or with expression of the AP2-like genes being regulated by a negative feedback loop.

A consensus genetic map of sorghum that integrates multiple component maps and high-throughput Diversity Array Technology (DArT) markers

Abstract: Background Sorghum genome mapping based on DNA markers began in the early 1990s and numerous genetic linkage maps of sorghum have been published in the last decade, based initially on RFLP markers with more recent maps including AFLPs and SSRs and very recently, Diversity Array Technology (DArT) markers. It is essential to integrate the rapidly growing body of genetic linkage data produced through DArT with the multiple genetic linkage maps for sorghum generated through other marker technologies. Here, we report on the colinearity of six independent sorghum component maps and on the integration of these component maps into a single reference resource that contains commonly utilized SSRs, AFLPs, and high-throughput DArT markers.

Arabidopsis WRKY2 transcription factor mediates seed germination and postgermination arrest of development by abscisic acid

Abstract: Plant WRKY DNA-binding transcription factors are key regulators in certain developmental programs. A number of studies have suggested that WRKY genes may mediate seed germination and postgermination growth. However, it is unclear whether WRKY genes mediate ABA-dependent seed germination and postgermination growth arrest. To determine directly the role of Arabidopsis WRKY2 transcription factor during ABA-dependent seed germination and postgermination growth arrest, we isolated T-DNA insertion mutants. Two independent T-DNA insertion mutants for WRKY2 were hypersensitive to ABA responses only during seed germination and postgermination early growth. wrky2 mutants displayed delayed or decreased expression of ABI5 and ABI3, but increased or prolonged expression of Em1 and Em6. wrky2 mutants and wild type showed similar levels of expression for miR159 and its target genes MYB33 and MYB101. Analysis of WRKY2 expression level in ABA-insensitive and ABA-deficient mutants abi5-1, abi3-1, aba2-3 and aba3-1 further indicated that ABA-induced WRKY2 accumulation during germination and postgermination early growth requires ABI5, ABI3, ABA2 and ABA3. ABA hypersensitivity of the wrky2 mutants during seed germination and postgermination early seedling establishment is attributable to elevated mRNA levels of ABI5, ABI3 and ABI5-induced Em1 and Em6 in the mutants. WRKY2-mediated ABA responses are independent of miR159 and its target genes MYB33 and MYB101. ABI5, ABI3, ABA2 and ABA3 are important regulators of the transcripts of WRKY2 by ABA treatment. Our results suggest that WRKY2 transcription factor mediates seed germination and postgermination developmental arrest by ABA.

Utility of EST-derived SSR in cultivated peanut (Arachis hypogaea L.) and Arachis wild species.

Abstract: Lack of sufficient molecular markers hinders current genetic research in peanuts (Arachis hypogaea L.). It is necessary to develop more molecular markers for potential use in peanut genetic research. With the development of peanut EST projects, a vast amount of available EST sequence data has been generated. These data offered an opportunity to identify SSR in ESTs by data mining. In this study, we investigated 24,238 ESTs for the identification and development of SSR markers. In total, 881 SSRs were identified from 780 SSR-containing unique ESTs. On an average, one SSR was found per 7.3 kb of EST sequence with tri-nucleotide motifs (63.9%) being the most abundant followed by di- (32.7%), tetra- (1.7%), hexa- (1.0%) and penta-nucleotide (0.7%) repeat types. The top six motifs included AG/TC (27.7%), AAG/TTC (17.4%), AAT/TTA (11.9%), ACC/TGG (7.72%), ACT/TGA (7.26%) and AT/TA (6.3%). Based on the 780 SSR-containing ESTs, a total of 290 primer pairs were successfully designed and used for validation of the amplification and assessment of the polymorphism among 22 genotypes of cultivated peanuts and 16 accessions of wild species. The results showed that 251 primer pairs yielded amplification products, of which 26 and 221 primer pairs exhibited polymorphism among the cultivated and wild species examined, respectively. Two to four alleles were found in cultivated peanuts, while 3–8 alleles presented in wild species. The apparent broad polymorphism was further confirmed by cloning and sequencing of amplified alleles. Sequence analysis of selected amplified alleles revealed that allelic diversity could be attributed mainly to differences in repeat type and length in the microsatellite regions. In addition, a few single base mutations were observed in the microsatellite flanking regions. This study gives an insight into the frequency, type and distribution of peanut EST-SSRs and demonstrates successful development of EST-SSR markers in cultivated peanut. These EST-SSR markers could enrich the current resource of molecular markers for the peanut community and would be useful for qualitative and quantitative trait mapping, marker-assisted selection, and genetic diversity studies in cultivated peanut as well as related Arachis species. All of the 251 working primer pairs with names, motifs, repeat types, primer sequences, and alleles tested in cultivated and wild species are listed in Additional File 1.

Identification and expression analysis of WRKY transcription factor genes in canola (Brassica napus L.) in response to fungal pathogens and hormone treatments.

Abstract: Background 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.

The cellulose synthase superfamily in fully sequenced plants and algae.

Abstract: The cellulose synthase superfamily has been classified into nine cellulose synthase-like (Csl) families and one cellulose synthase (CesA) family. The Csl families have been proposed to be involved in the synthesis of the backbones of hemicelluloses of plant cell walls. With 17 plant and algal genomes fully sequenced, we sought to conduct a genome-wide and systematic investigation of this superfamily through in-depth phylogenetic analyses. A single-copy gene is found in the six chlorophyte green algae, which is most closely related to the CslA and CslC families that are present in the seven land plants investigated in our analyses. Six proteins from poplar, grape and sorghum form a distinct family (CslJ), providing further support for the conclusions from two recent studies. CslB/E/G/H/J families have evolved significantly more rapidly than their widely distributed relatives, and tend to have intragenomic duplications, in particular in the grape genome. Our data suggest that the CslA and CslC families originated through an ancient gene duplication event in land plants. We speculate that the single-copy Csl gene in green algae may encode a mannan synthase. We confirm that the rest of the Csl families have a different evolutionary origin than CslA and CslC, and have proposed a model for the divergence order among them. Our study provides new insights about the evolution of this important gene family in plants.

Development of SSR markers and analysis of diversity in Turkish populations of Brachypodium distachyon

Abstract: Brachypodium distachyon (Brachypodium) is rapidly emerging as a powerful model system to facilitate research aimed at improving grass crops for grain, forage and energy production. To characterize the natural diversity of Brachypodium and provide a valuable new tool to the growing list of resources available to Brachypodium researchers, we created and characterized a large, diverse collection of inbred lines. We developed 84 inbred lines from eight locations in Turkey. To enable genotypic characterization of this collection, we created 398 SSR markers from BAC end and EST sequences. An analysis of 187 diploid lines from 56 locations with 43 SSR markers showed considerable genotypic diversity. There was some correlation between SSR genotypes and broad geographic regions, but there was also a high level of genotypic diversity at individual locations. Phenotypic analysis of this new germplasm resource revealed considerable variation in flowering time, seed size, and plant architecture. The inbreeding nature of Brachypodium was confirmed by an extremely high level of homozygosity in wild plants and a lack of cross-pollination under laboratory conditions. Taken together, the inbreeding nature and genotypic diversity observed at individual locations suggest a significant amount of long-distance seed dispersal. The resources developed in this study are freely available to the research community and will facilitate experimental applications based on natural diversity.

Characterization of microsatellites and gene contents from genome shotgun sequences of mungbean (Vigna radiata (L.) Wilczek).

Abstract: Mungbean is an important economical crop in Asia. However, genomic research has lagged behind other crop species due to the lack of polymorphic DNA markers found in this crop. The objective of this work is to develop and characterize microsatellite or simple sequence repeat (SSR) markers from genome shotgun sequencing of mungbean. We have generated and characterized a total of 470,024 genome shotgun sequences covering 100.5 Mb of the mungbean (Vigna radiata (L.) Wilczek) genome using 454 sequencing technology. We identified 1,493 SSR motifs that could be used as potential molecular markers. Among 192 tested primer pairs in 17 mungbean accessions, 60 loci revealed polymorphism with polymorphic information content (PIC) values ranging from 0.0555 to 0.6907 with an average of 0.2594. Majority of microsatellite markers were transferable in Vigna species, whereas transferability rates were only 22.90% and 24.43% in Phaseolus vulgaris and Glycine max, respectively. We also used 16 SSR loci to evaluate phylogenetic relationship of 35 genotypes of the Asian Vigna group. The genome survey sequences were further analyzed to search for gene content. The evidence suggested 1,542 gene fragments have been sequence tagged, that fell within intersected existing gene models and shared sequence homology with other proteins in the database. Furthermore, potential microRNAs that could regulate developmental stages and environmental responses were discovered from this dataset. In this report, we provided evidence of generating remarkable levels of diverse microsatellite markers and gene content from high throughput genome shotgun sequencing of the mungbean genomic DNA. The markers could be used in germplasm analysis, accessing genetic diversity and linkage mapping of mungbean.

Characterization of WRKY co-regulatory networks in rice and Arabidopsis

Abstract: The WRKY transcription factor gene family has a very ancient origin and has undergone extensive duplications in the plant kingdom. Several studies have pointed out their involvement in a range of biological processes, revealing that a large number of WRKY genes are transcriptionally regulated under conditions of biotic and/or abiotic stress. To investigate the existence of WRKY co-regulatory networks in plants, a whole gene family WRKYs expression study was carried out in rice (Oryza sativa). This analysis was extended to Arabidopsis thaliana taking advantage of an extensive repository of gene expression data. The presented results suggested that 24 members of the rice WRKY gene family (22% of the total) were differentially-regulated in response to at least one of the stress conditions tested. We defined the existence of nine OsWRKY gene clusters comprising both phylogenetically related and unrelated genes that were significantly co-expressed, suggesting that specific sets of WRKY genes might act in co-regulatory networks. This hypothesis was tested by Pearson Correlation Coefficient analysis of the Arabidopsis WRKY gene family in a large set of Affymetrix microarray experiments. AtWRKYs were found to belong to two main co-regulatory networks (COR-A, COR-B) and two smaller ones (COR-C and COR-D), all including genes belonging to distinct phylogenetic groups. The COR-A network contained several AtWRKY genes known to be involved mostly in response to pathogens, whose physical and/or genetic interaction was experimentally proven. We also showed that specific co-regulatory networks were conserved between the two model species by identifying Arabidopsis orthologs of the co-expressed OsWRKY genes. In this work we identified sets of co-expressed WRKY genes in both rice and Arabidopsis that are functionally likely to cooperate in the same signal transduction pathways. We propose that, making use of data from co-regulatory networks, it is possible to highlight novel clusters of plant genes contributing to the same biological processes or signal transduction pathways. Our approach will contribute to unveil gene cooperation pathways not yet identified by classical genetic analyses. This information will open new routes contributing to the dissection of WRKY signal transduction pathways in plants.

The cinnamyl alcohol dehydrogenase gene family in Populus : phylogeny, organization, and expression

Abstract: Lignin is a phenolic heteropolymer in secondary cell walls that plays a major role in the development of plants and their defense against pathogens. The biosynthesis of monolignols, which represent the main component of lignin involves many enzymes. The cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis as it catalyzes the final step in the synthesis of monolignols. The CAD gene family has been studied in Arabidopsis thaliana, Oryza sativa and partially in Populus. This is the first comprehensive study on the CAD gene family in woody plants including genome organization, gene structure, phylogeny across land plant lineages, and expression profiling in Populus. The phylogenetic analyses showed that CAD genes fall into three main classes (clades), one of which is represented by CAD sequences from gymnosperms and angiosperms. The other two clades are represented by sequences only from angiosperms. All Populus CAD genes, except PoptrCAD 4 are distributed in Class II and Class III. CAD genes associated with xylem development (PoptrCAD 4 and PoptrCAD 10) belong to Class I and Class II. Most of the CAD genes are physically distributed on duplicated blocks and are still in conserved locations on the homeologous duplicated blocks. Promoter analysis of CAD genes revealed several motifs involved in gene expression modulation under various biological and physiological processes. The CAD genes showed different expression patterns in poplar with only two genes preferentially expressed in xylem tissues during lignin biosynthesis. The phylogeny of CAD genes suggests that the radiation of this gene family may have occurred in the early ancestry of angiosperms. Gene distribution on the chromosomes of Populus showed that both large scale and tandem duplications contributed significantly to the CAD gene family expansion. The duplication of several CAD genes seems to be associated with a genome duplication event that happened in the ancestor of Salicaceae. Phylogenetic analyses associated with expression profiling and results from previous studies suggest that CAD genes involved in wood development belong to Class I and Class II. The other CAD genes from Class II and Class III may function in plant tissues under biotic stresses. The conservation of most duplicated CAD genes, the differential distribution of motifs in their promoter regions, and the divergence of their expression profiles in various tissues of Populus plants indicate that genes in the CAD family have evolved tissue-specialized expression profiles and may have divergent functions.

Selection of reference genes for quantitative real-time PCR expression studies in the apomictic and sexual grass Brachiaria brizantha

Abstract: Brachiaria brizantha is an important forage grass. The occurrence of both apomictic and sexual reproduction within Brachiaria makes it an interesting system for understanding the molecular pathways involved in both modes of reproduction. Quantitative real time PCR (qRT-PCR) has emerged as an important technique to compare expression profile of target genes and, in order to obtain reliable results, it is important to have suitable reference genes. In this work, we evaluated eight potential reference genes for B. brizantha qRT-PCR experiments, isolated from cDNA ovary libraries. Vegetative and reproductive tissues of apomictic and sexual B. brizantha were tested to validate the reference genes, including the female gametophyte, where differences in the expression profile between sexual and apomictic plants must occur.

Vitamin B6 deficient plants display increased sensitivity to high light and photo-oxidative stress

Abstract: Background Vitamin B6 is a collective term for a group of six interconvertible compounds: pyridoxine, pyridoxal, pyridoxamine and their phosphorylated derivatives. Vitamin B6 plays essential roles as a cofactor in a range of biochemical reactions. In addition, vitamin B6 is able to quench reactive oxygen species in vitro, and exogenously applied vitamin B6 protects plant cells against cell death induced by singlet oxygen (1O2). These results raise the important question as to whether plants employ vitamin B6 as an antioxidant to protect themselves against reactive oxygen species.

CO2 assimilation, ribulose-1,5-bisphosphate carboxylase/oxygenase, carbohydrates and photosynthetic electron transport probed by the JIP-test, of tea leaves in response to phosphorus supply

Abstract: Although the effects of P deficiency on tea (Camellia sinensis (L.) O. Kuntze) growth, P uptake and utilization as well as leaf gas exchange and Chl a fluorescence have been investigated, very little is known about the effects of P deficiency on photosynthetic electron transport, photosynthetic enzymes and carbohydrates of tea leaves. In this study, own-rooted 10-month-old tea trees were supplied three times weekly for 17 weeks with 500 mL of nutrient solution at a P concentration of 0, 40, 80, 160, 400 or 1000 μM. This objective of this study was to determine how P deficiency affects CO2 assimilation, Rubisco, carbohydrates and photosynthetic electron transport in tea leaves to understand the mechanism by which P deficiency leads to a decrease in CO2 assimilation. Both root and shoot dry weight increased as P supply increased from 0 to 160 μM, then remained unchanged. P-deficient leaves from 0 to 80 μM P-treated trees showed decreased CO2 assimilation and stomatal conductance, but increased intercellular CO2 concentration. Both initial and total Rubisco activity, contents of Chl and total soluble protein in P-deficient leaves decreased to a lesser extent than CO2 assimilation. Contents of sucrose and starch were decreased in P-deficient leaves, whereas contents of glucose and fructose did not change significantly except for a significant increase in the lowest P leaves. OJIP transients from P-deficient leaves displayed a rise at the O-step and a depression at the P-step, accompanied by two new steps at about 150 μs (L-step) and at about 300 μs (K-step). RC/CSo, TRo/ABS (or Fv/Fm), ETo/ABS, REo/ABS, maximum amplitude of IP phase, PIabs and PItot, abs were decreased in P-deficient leaves, while VJ, VI and dissipated energy were increased. P deficiency decreased photosynthetic electron transport capacity by impairing the whole electron transport chain from the PSII donor side up to the PSI, thus decreasing ATP content which limits RuBP regeneration, and hence, the rate of CO2 assimilation. Energy dissipation is enhanced to protect P-deficient leaves from photo-oxidative damage in high light.

Identification of candidate genome regions controlling disease resistance in Arachis.

Abstract: Background: Worldwide, diseases are important reducers of peanut (Arachis hypogaea) yield. Sources of resistance against many diseases are available in cultivated peanut genotypes, although often not in farmer preferred varieties. Wild species generally harbor greater levels of resistance and even apparent immunity, although the linkage of agronomically un-adapted wild alleles with wild disease resistance genes is inevitable. Marker-assisted selection has the potential to facilitate the combination of both cultivated and wild resistance loci with agronomically adapted alleles. However, in peanut there is an almost complete lack of knowledge of the regions of the Arachis genome that control disease resistance. Results: In this work we identified candidate genome regions that control disease resistance. For this we placed candidate disease resistance genes and QTLs against late leaf spot disease on the genetic map of the A-genome of Arachis, which is based on microsatellite markers and legume anchor markers. These marker types are transferable within the genus Arachis and to other legumes respectively, enabling this map to be aligned to other Arachis maps and to maps of other legume crops including those with sequenced genomes. In total, 34 sequence-confirmed candidate disease resistance genes and five QTLs were mapped. Conclusion: Candidate genes and QTLs were distributed on all linkage groups except for the smallest, but the distribution was not even. Groupings of candidate genes and QTLs for late leaf spot resistance were apparent on the upper region of linkage group 4 and the lower region of linkage group 2, indicating that these regions are likely to control disease resistance.

Identification, characterization and utilization of unigene derived microsatellite markers in tea (Camellia sinensis L.)

Abstract: Despite great advances in genomic technology observed in several crop species, the availability of molecular tools such as microsatellite markers has been limited in tea (Camellia sinensis L.). The development of microsatellite markers will have a major impact on genetic analysis, gene mapping and marker assisted breeding. Unigene derived microsatellite (UGMS) markers identified from publicly available sequence database have the advantage of assaying variation in the expressed component of the genome with unique identity and position. Therefore, they can serve as efficient and cost effective alternative markers in such species. Considering the multiple advantages of UGMS markers, 1,223 unigenes were predicted from 2,181 expressed sequence tags (ESTs) of tea (Camellia sinensis L.). A total of 109 (8.9%) unigenes containing 120 SSRs were identified. SSR abundance was one in every 3.55 kb of EST sequences. The microsatellites mainly comprised of di (50.8%), tri (30.8%), tetra (6.6%), penta (7.5%) and few hexa (4.1%) nucleotide repeats. Among the dinucleotide repeats, (GA)n.(TC)n were most abundant (83.6%). Ninety six primer pairs could be designed form 83.5% of SSR containing unigenes. Of these, 61 (63.5%) primer pairs were experimentally validated and used to investigate the genetic diversity among the 34 accessions of different Camellia spp. Fifty one primer pairs (83.6%) were successfully cross transferred to the related species at various levels. Functional annotation of the unigenes containing SSRs was done through gene ontology (GO) characterization. Thirty six (60%) of them revealed significant sequence similarity with the known/putative proteins of Arabidopsis thaliana. Polymorphism information content (PIC) ranged from 0.018 to 0.972 with a mean value of 0.497. The average heterozygosity expected (H E ) and observed (H o ) obtained was 0.654 and 0.413 respectively, thereby suggesting highly heterogeneous nature of tea. Further, test for IAM and SMM models for the UGMS loci showed excess heterozygosity and did not show any bottleneck operating in the tea population. UGMS markers identified and characterized in this study provided insight about the abundance and distribution of SSR in the expressed genome of C. sinensis. The identification and validation of 61 new UGMS markers will not only help in intra and inter specific genetic diversity assessment but also be enriching limited microsatellite markers resource in tea. Further, the use of these markers would reduce the cost and facilitate the gene mapping and marker-aided selection in tea. Since, 36 of these UGMS markers correspond to the Arabidopsis protein sequence data with known functions will offer the opportunity to investigate the consequences of SSR polymorphism on gene functions.

Removing celiac disease-related gluten proteins from bread wheat while retaining technological properties: a study with Chinese Spring deletion lines

Abstract: Gluten proteins can induce celiac disease (CD) in genetically susceptible individuals. In CD patients gluten-derived peptides are presented to the immune system, which leads to a CD4+ T-cell mediated immune response and inflammation of the small intestine. However, not all gluten proteins contain T-cell stimulatory epitopes. Gluten proteins are encoded by multigene loci present on chromosomes 1 and 6 of the three different genomes of hexaploid bread wheat (Triticum aestivum) (AABBDD). The effects of deleting individual gluten loci on both the level of T-cell stimulatory epitopes in the gluten proteome and the technological properties of the flour were analyzed using a set of deletion lines of Triticum aestivum cv. Chinese Spring. The reduction of T-cell stimulatory epitopes was analyzed using monoclonal antibodies that recognize T-cell epitopes present in gluten proteins. The deletion lines were technologically tested with respect to dough mixing properties and dough rheology. The results show that removing the α-gliadin locus from the short arm of chromosome 6 of the D-genome (6DS) resulted in a significant decrease in the presence of T-cell stimulatory epitopes but also in a significant loss of technological properties. However, removing the ω-gliadin, γ-gliadin, and LMW-GS loci from the short arm of chromosome 1 of the D-genome (1DS) removed T-cell stimulatory epitopes from the proteome while maintaining technological properties. The consequences of these data are discussed with regard to reducing the load of T-cell stimulatory epitopes in wheat, and to contributing to the design of CD-safe wheat varieties.

New sources of soybean seed meal and oil composition traits identified through TILLING.

Abstract: Background Several techniques are available to study gene function, but many are less than ideal for soybean. Reverse genetics, a relatively new approach, can be utilized to identify novel mutations in candidate genes; this technique has not produced an allelic variant with a confirmed phenotype in soybean. Soybean raffinose synthase genes and microsomal omega-6 fatty acid desaturase genes were screened for novel alleles in mutagenized soybean populations.

Genetic mapping of wild introgressions into cultivated peanut: a way toward enlarging the genetic basis of a recent allotetraploid.

Abstract: Background Peanut (Arachis hypogaea L.) is widely used as a food and cash crop around the world. It is considered to be an allotetraploid (2n = 4x = 40) originated from a single hybridization event between two wild diploids. The most probable hypothesis gave A. duranensis as the wild donor of the A genome and A. ipaensis as the wild donor of the B genome. A low level of molecular polymorphism is found in cultivated germplasm and up to date few genetic linkage maps have been published. The utilization of wild germplasm in breeding programs has received little attention due to the reproductive barriers between wild and cultivated species and to the technical difficulties encountered in making large number of crosses. We report here the development of a SSR based genetic map and the analysis of genome-wide segment introgressions into the background of a cultivated variety through the utilization of a synthetic amphidiploid between A. duranensis and A. ipaensis.

A set of EST-SNPs for map saturation and cultivar identification in melon

Abstract: Background There are few genomic tools available in melon (Cucumis melo L.), a member of the Cucurbitaceae, despite its importance as a crop. Among these tools, genetic maps have been constructed mainly using marker types such as simple sequence repeats (SSR), restriction fragment length polymorphisms (RFLP) and amplified fragment length polymorphisms (AFLP) in different mapping populations. There is a growing need for saturating the genetic map with single nucleotide polymorphisms (SNP), more amenable for high throughput analysis, especially if these markers are located in gene coding regions, to provide functional markers. Expressed sequence tags (ESTs) from melon are available in public databases, and resequencing ESTs or validating SNPs detected in silico are excellent ways to discover SNPs.

Bioaccumulation and toxicity of selenium compounds in the green alga Scenedesmus quadricauda

Abstract: Selenium is a trace element performing important biological functions in many organisms including humans. It usually affects organisms in a strictly dosage-dependent manner being essential at low and toxic at higher concentrations. The impact of selenium on mammalian and land plant cells has been quite extensively studied. Information about algal cells is rare despite of the fact that they could produce selenium enriched biomass for biotechnology purposes. We studied the impact of selenium compounds on the green chlorococcal alga Scenedesmus quadricauda. Both the dose and chemical forms of Se were critical factors in the cellular response. Se toxicity increased in cultures grown under sulfur deficient conditions. We selected three strains of Scenedesmus quadricauda specifically resistant to high concentrations of inorganic selenium added as selenite (Na2SeO3) – strain SeIV, selenate (Na2SeO4) – strain SeVI or both – strain SeIV+VI. The total amount of Se and selenomethionine in biomass increased with increasing concentration of Se in the culturing media. The selenomethionine made up 30–40% of the total Se in biomass. In both the wild type and Se-resistant strains, the activity of thioredoxin reductase, increased rapidly in the presence of the form of selenium for which the given algal strain was not resistant. The selenium effect on the green alga Scenedesmus quadricauda was not only dose dependent, but the chemical form of the element was also crucial. With sulfur deficiency, the selenium toxicity increases, indicating interference of Se with sulfur metabolism. The amount of selenium and SeMet in algal biomass was dependent on both the type of compound and its dose. The activity of thioredoxin reductase was affected by selenium treatment in dose-dependent and toxic-dependent manner. The findings implied that the increase in TR activity in algal cells was a stress response to selenium cytotoxicity. Our study provides a new insight into the impact of selenium on green algae, especially with regard to its toxicity and bioaccumulation.

A linkage map for the B-genome of Arachis (Fabaceae) and its synteny to the A-genome.

Abstract: Background Arachis hypogaea (peanut) is an important crop worldwide, being mostly used for edible oil production, direct consumption and animal feed Cultivated peanut is an allotetraploid species with two different genome components, A and B Genetic linkage maps can greatly assist molecular breeding and genomic studies However, the development of linkage maps for A hypogaea is difficult because it has very low levels of polymorphism This can be overcome by the utilization of wild species of Arachis, which present the A- and B-genomes in the diploid state, and show high levels of genetic variability

Mapping quantitative trait loci (QTLs) for fatty acid composition in an interspecific cross of oil palm

Abstract: Marker Assisted Selection (MAS) is well suited to a perennial crop like oil palm, in which the economic products are not produced until several years after planting. The use of DNA markers for selection in such crops can greatly reduce the number of breeding cycles needed. With the use of DNA markers, informed decisions can be made at the nursery stage, regarding which individuals should be retained as breeding stock, which are satisfactory for agricultural production, and which should be culled. The trait associated with oil quality, measured in terms of its fatty acid composition, is an important agronomic trait that can eventually be tracked using molecular markers. This will speed up the production of new and improved oil palm planting materials. A map was constructed using AFLP, RFLP and SSR markers for an interspecific cross involving a Colombian Elaeis oleifera (UP1026) and a Nigerian E. guinneensis (T128). A framework map was generated for the male parent, T128, using Joinmap ver. 4.0. In the paternal (E. guineensis) map, 252 markers (199 AFLP, 38 RFLP and 15 SSR) could be ordered in 21 linkage groups (1815 cM). Interval mapping and multiple-QTL model (MQM) mapping (also known as composite interval mapping, CIM) were used to detect quantitative trait loci (QTLs) controlling oil quality (measured in terms of iodine value and fatty acid composition). At a 5% genome-wide significance threshold level, QTLs associated with iodine value (IV), myristic acid (C14:0), palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), oleic acid (C18:1) and linoleic acid (C18:2) content were detected. One genomic region on Group 1 appears to be influencing IV, C14:0, C16:0, C18:0 and C18:1 content. Significant QTL for C14:0, C16:1, C18:0 and C18:1 content was detected around the same locus on Group 15, thus revealing another major locus influencing fatty acid composition in oil palm. Additional QTL for C18:0 was detected on Group 3. A minor QTL for C18:2 was detected on Group 2. This study describes the first successful detection of QTLs for fatty acid composition in oil palm. These QTLs constitute useful tools for application in breeding programmes.

Gene-based microsatellites for cassava (Manihot esculenta Crantz): prevalence, polymorphisms, and cross-taxa utility

Abstract: Cassava (Manihot esculenta Crantz), a starchy root crop grown in tropical and subtropical climates, is the sixth most important crop in the world after wheat, rice, maize, potato and barley. The repertoire of simple sequence repeat (SSR) markers for cassava is limited and warrants a need for a larger number of polymorphic SSRs for germplasm characterization and breeding applications. A total of 846 putative microsatellites were identified in silico from an 8,577 cassava unigene set with an average density of one SSR every 7 kb. One hundred and ninety-two candidate SSRs were screened for polymorphism among a panel of cassava cultivars from Africa, Latin America and Asia, four wild Manihot species as well as two other important taxa in the Euphorbiaceae, leafy spurge (Euphorbia esula) and castor bean (Ricinus communis). Of 168 markers with clean amplification products, 124 (73.8%) displayed polymorphism based on high resolution agarose gels. Of 85 EST-SSR markers screened, 80 (94.1%) amplified alleles from one or more wild species (M epruinosa, M glaziovii, M brachyandra, M tripartita) whereas 13 (15.3%) amplified alleles from castor bean and 9 (10.6%) amplified alleles from leafy spurge; hence nearly all markers were transferable to wild relatives of M esculenta while only a fraction was transferable to the more distantly related taxa. In a subset of 20 EST-SSRs assessed by fluorescence-based genotyping the number of alleles per locus ranged from 2 to 10 with an average of 4.55 per locus. These markers had a polymorphism information content (PIC) from 0.19 to 0.75 with an average value of 0.55 and showed genetic relationships consistent with existing information on these genotypes. A set of 124 new, unique polymorphic EST-SSRs was developed and characterized which extends the repertoire of SSR markers for cultivated cassava and its wild relatives. The markers show high PIC values and therefore will be useful for cultivar identification, taxonomic studies, and genetic mapping. The study further shows that mining ESTs is a highly efficient strategy for polymorphism detection within the cultivated cassava gene pool.