Gene expression profiles of arabidopsis under the stress of methyl viologen: a microarray analysis

Methyl viologen (MV) is the main ingredient of Paraquat. It is little known about how plants respond to this compound. To understand the mode of MV action and molecular mechanism of plant response, we performed experiments of microarray on Arabidopsis. In MV treated seedling, approximately 6 % genes were altered at mRNA levels, including 818 genes increased, whereas 1,440 genes decreased. Studies of these genes expression patterns provided some new information on the reaction process of plant after the treatment with MV. These included signaling molecules for MV response and reactive oxygen species formation, enzymes required for secondary metabolism and, cell wall maintenance and strategy of photostasis balance. The expression kinetics of the genes induced by MV will provides useful information for the abiotic stress defense mechanism in plants.     

Genomic Inventory and Transcriptional Analysis of Medicago truncatula Transporters

Transporters move hydrophilic substrates across hydrophobic biological membranes and play key roles in plant nutrition, metabolism, and signaling and, consequently, in plant growth, development, and responses to the environment. To initiate and support systematic characterization of transporters in the model legume Medicago truncatula, we identified 3,830 transporters and classified 2,673 of these into 113 families and 146 subfamilies. Analysis of gene expression data for 2,611 of these transporters identified 129 that are expressed in an organ-specific manner, including 50 that are nodule specific and 36 specific to mycorrhizal roots. Further analysis uncovered 196 transporters that are induced at least 5-fold during nodule development and 44 in roots during arbuscular mycorrhizal symbiosis. Among the nodule- and mycorrhiza-induced transporter genes are many candidates for known transport activities in these beneficial symbioses. The data presented here are a unique resource for the selection and functional characterization of legume transporters.Transporters are membrane-spanning proteins that selectively transport hydrophilic solutes across hydrophobic membranes. They are present and required in all cellular membranes, including the cell or plasma membrane that separates cellular contents from the external environment and membranes of the various subcellular organelles. By transporting metabolites and nonmetabolites, such as inorganic ions, transporters play integral roles in cell metabolism, ion homeostasis, osmoregulation, signaling, and other processes. Transporters move solutes not only within cells but also between cells, tissues, and organs of complex, multicellular organisms such as higher plants. Therefore, they help to coordinate metabolic, physiological, and developmental processes in higher plants and other organisms.Transporter proteins/complexes contain multiple membrane-spanning domains that form an aqueous pore in the membrane, which enables movement of selected solutes from one side of the membrane to the other. Membrane-spanning domains are hydrophobic in nature, or at least partially so, which enables them to interact with the phospholipid bilayer of membranes. Many transporters contain hydrophobic α -helical segments that span the membrane, while others contain β -barrel transmembrane domains (TMD). Computer programs have been developed to identify putative membrane-spanning α -helices (Hoffman and Stoffel, 1993; Hirokawa et al., 1998; Tusnady and Simon, 2001) and β -barrels (Koebnik et al., 2000; Valavanis et al., 2006), which facilitate de novo prediction of putative membrane proteins, including transporters. Databases of known, characterized transport proteins aid in the identification and classification of transporters in new species via sequence similarity. Perhaps the most comprehensive of these is the Transporter Classification Database (TCDB; Saier et al., 2006), which was created to serve as a repository of functionally characterized transporters. It also serves to categorize new transporters into families and subfamilies based on molecular, evolutionary, and functional properties. At present, it consists of approximately 3,000 transporters classified in more than 500 families (www.tcdb.org).The legume family is second only to the grass family in importance to humans as a source of food, feed for livestock, and raw materials for industry (Graham and Vance, 2003). Legumes are the lynch pin of sustainable agriculture, because they supply their own nitrogen by “fixing” it (reducing N₂ to NH₃) in a symbiotic association with bacteria called rhizobia. This mutually beneficial association provides legumes and subsequent crops with a free and renewable source of usable nitrogen (Udvardi and Day, 1997). Legumes also establish symbiosis with mycorrhizal fungi that help the plant mine phosphorous and other nutrients from the soil (Smith and Read, 2008).Symbiotic nitrogen fixation (SNF) in root nodule cells of legumes is carried out by rhizobia that are completely surrounded by a plant membrane called the symbiosome membrane (SM), which forms a nitrogen-fixing organelle, the symbiosome, within the plant cytoplasm. Infected cortical cells of nodules contain thousands of symbiosomes, each containing one or a few bacteria. Infected plant cells, interspersed with noninfected cells, constitute the central tissue of nodules, which is surrounded by uninfected tissue that restricts gas exchange with the soil, and phloem and xylem, which import and export nutrients from the nodule, respectively. In exchange for ammonium produced by bacterial nitrogenase and released to the plant, rhizobia receive reduced carbon (principally dicarboxylic acids such as malate) and every other nutrient required for bacterial cell growth and maintenance (Udvardi and Day, 1997). Exchange of nutrients between the plant cell cytoplasm and rhizobia is mediated by a variety of transporters in the SM, some of which are induced during nodule development (Benedito et al., 2008). Transporters perform many other important roles in nodules, such as short- and long-distance transport of nutrients between plant cells and tissues and between the nodule and other organs, processes facilitated by proteins of the plant cell plasma membrane. On the other hand, transporters on the membranes of organelles such as mitochondria, plastids, and peroxisomes facilitate the movement of metabolites between cellular compartments, which is crucial for nodule metabolism and SNF.In the arbuscular mycorrhizal (AM) symbiosis, the fungal symbionts inhabit the root cortex, where they obtain carbon from the plant, and in exchange they deliver mineral nutrients, particularly phosphorus and nitrogen, to the root. Mineral nutrient transfer between symbionts occurs at a specialized symbiotic interface between branched hyphae, called arbuscules, and the cortical cells that they inhabit (Parniske, 2008). The interface is delimited by a plant-derived membrane called the periarbuscular membrane, which is continuous with the plasma membrane but contains some unique proteins, including novel inorganic phosphate (Pi) transporters (Harrison et al., 2002; Paszkowski et al., 2002). These transporters are required to transfer Pi that is released from the arbuscule into the cortical cell. It is assumed, but not yet shown directly, that nitrogen, and possibly other mineral nutrients such as zinc, is also transferred between the symbionts at this membrane interface (Smith and Read, 2008). However, the transport proteins involved are currently unknown. Likewise, transporters involved in carbon transfer to the fungal symbiont have not been identified. While it is expected that the periarbuscular membrane will contain additional transport activities, only a handful of transporters residing in this membrane have been identified to date.Although inroads have been made in the characterization of individual transporters in a variety of legume species, no systematic work has been done to identify and characterize all the transporters in any one species. Three legume species, Medicago truncatula, Glycine max (soybean), and Lotus japonicus, have been the subject of extensive cDNA and genomic DNA sequencing over the past few years (Young et al., 2003, 2005; Sato et al., 2007, 2008), making them interesting model systems for whole-genome analysis of transporters. The genome sequence of M. truncatula is being annotated by the International Medicago Genome Annotation Group (IMGAG), which described 38,335 genes in its version 2.0 of the genome sequence (http://www.medicago.org/genome/downloads/Mt2/). Additional resources relevant to Medicago functional genomics include the Medicago Gene Expression Atlas (http://bioinfo.noble.org/gene-atlas/v2), which provides developmental expression data for the majority of Medicago genes (Benedito et al., 2008), and a Tnt1 transposon-insertion mutant population with insertions in the majority of genes, which enables efficient forward and reverse genetics (Tadege et al., 2005, 2008). To facilitate systematic functional analysis of transporters in Medicago, and especially those involved in nitrogen-fixing and AM symbioses, we have identified and categorized 2,673 transporter genes and analyzed the expression patterns of 2,604 of these. The results of this work are presented here.     

Integrating Constitutive Gene Expression and Chemoactivity: Mining the NCI60 Anticancer Screen

Studies into the genetic origins of tumor cell chemoactivity pose significant challenges to bioinformatic mining efforts. Connections between measures of gene expression and chemoactivity have the potential to identify clinical biomarkers of compound response, cellular pathways important to efficacy and potential toxicities; all vital to anticancer drug development. An investigation has been conducted that jointly explores tumor-cell constitutive NCI60 gene expression profiles and small-molecule NCI60 growth inhibition chemoactivity profiles, viewed from novel applications of self-organizing maps (SOMs) and pathway-centric analyses of gene expressions, to identify subsets of over- and under-expressed pathway genes that discriminate chemo-sensitive and chemo-insensitive tumor cell types. Linear Discriminant Analysis (LDA) is used to quantify the accuracy of discriminating genes to predict tumor cell chemoactivity. LDA results find 15% higher prediction accuracies, using ∼30% fewer genes, for pathway-derived discriminating genes when compared to genes derived using conventional gene expression-chemoactivity correlations. The proposed pathway-centric data mining procedure was used to derive discriminating genes for ten well-known compounds. Discriminating genes were further evaluated using gene set enrichment analysis (GSEA) to reveal a cellular genetic landscape, comprised of small numbers of key over and under expressed on- and off-target pathway genes, as important for a compound’s tumor cell chemoactivity. Literature-based validations are provided as support for chemo-important pathways derived from this procedure. Qualitatively similar results are found when using gene expression measurements derived from different microarray platforms. The data used in this analysis is available at http://pubchem.ncbi.nlm.nih.gov/and http://www.ncbi.nlm.nih.gov/projects/geo ({"type":"entrez-geo","attrs":{"text":"GPL96","term_id":"96"}}GPL96, {"type":"entrez-geo","attrs":{"text":"GSE32474","term_id":"32474"}}GSE32474).     

Overexpression of phytosulfokine-α induces male sterility and cell growth by regulating cell wall development in Arabidopsis

Key message

Over-production of functional PSK-α in Arabidopsis caused increases in both plant cell growth and biomass and induced male sterility by regulating cell wall development.

Abstract

Phytosulfokine-α (PSK-α) is a novel disulfated pentapeptide hormone that is involved in promoting plant cell growth. Although a role for PSK-α in stimulating protoplast expansion has been suggested, how PSK-α regulates cell growth in planta remains poorly understood. In this study, we found that overexpression of the normal PSK-α precursor gene AtPSK4, which resulted in high levels of PSK-α, caused longer roots and larger leaves with enlarged cells. As expected, these changes were not observed in transgenic plants overexpressing mutated AtPSK4, which generated unsulfated PSK-α. These findings confirmed the role of PSK-α in promoting plant cell growth. Furthermore, we found that overexpressing AtPSK4, but not mutated AtPSK4, induced a phenotype of male sterility that resulted from the failure of fibrous cell wall development in the endothecium. In addition, overexpressing AtPSK4 enhanced expression of a number of genes encoding expansins, which are involved in cell wall loosening. Accordingly, in addition to its role in cell growth, we propose a novel function for PSK-α signaling in the modulation of plant male sterility via regulation of cell wall development.

     

Mutation in the Arabidopsis PASTICCINO1 Gene,Which Encodes a New FK506-Binding Protein-Like Protein,Has a Dramatic Effect on Plant Development

The pasticcino (pas) mutants of Arabidopsis thaliana are a new class of plant developmental mutants; members of this class show ectopic cell proliferation in cotyledons, extra layers of cells in the hypocotyl, and an abnormal apical meristem. This phenotype is correlated with both cell division and cell elongation defects. There are three complementation groups of pas mutants (pas1, pas2, and pas3, with, respectively 2, 1, and 4 alleles). Here we describe in more detail the pas1-1 allele, which was obtained by insertional mutagenesis. The PAS1 gene has been cloned and characterized; it encodes an immunophilin-like protein similar to the p59 FK506-binding protein (FKBP52). PAS1 is characterized by an FKBP-like domain and three tetratricopeptide repeat units. Although the presence of immunophilins in plants has already been demonstrated, the pas1-1 mutant represents the first inactivation of an FKBP-like gene in plants. PAS1 expression is altered in pas1 mutants and in the pas2 and pas3 mutants. The expression of the PAS1 gene is increased in the presence of cytokinins, a class of phytohormones originally discovered because of their ability to stimulate cell division. These results are of particular relevance as they show for the first time that an FKBP-like protein plays an important role in the control of plant development.In flowering plants, morphogenesis depends on the control of the pattern and numbers of cell divisions and on the control of cell elongation. Although there are many examples of controlled patterns of cell division, we still know very little about how local patterns of cell division are established and maintained (30). In Arabidopsis thaliana, the roles of cell division control in the development of the embryo, the shoot, and the root have been extensively studied (reviewed in references 29 and 30). In the last few years, much progress has been made in this field by the isolation of mutants in which single-gene mutations affect specific modes of cell division control. Some of the corresponding genes have been cloned from A. thaliana (SHOOT MERISTEMLESS [STM] and SCARECROW [SCR]) maize (KNOTTED1), and petunia (NO APICAL MERISTEM) (reviewed in reference 30). These genes do not seem to specify components of the cell division machinery, but they are thought to act upstream in the control of cell division. The elements at the interface between genes like STM and SCR and cell cycle regulators, such as cyclins and the CDC genes, are still unknown.The growth and differentiation of higher plants is also greatly dependent on environmental stimuli, such as light and temperature, and on endogenous factors, such as phytohormones. Cytokinins (CKs) were originally discovered because of their ability to promote, along with auxins, plant cell division and organogenesis (reviewed in reference 9). Although this discovery initiated a vast amount of fundamental and applied research on the hormonal control of cell proliferation and regeneration, the mechanisms by which auxins and CKs act and interact at the molecular level are unknown. Steroid-like plant growth factors termed brassinosteroids (BR) were first characterized as inducing cell elongation in synergy with auxin, but recently these hormones have also been found to control plant cell divisions and morphogenesis (15; reviewed in reference 11).The genetic and molecular analysis of hormonal mutants is proving to be a powerful tool for unraveling the mode of action of these molecules. In an attempt to understand the mode of action of CKs and their molecular relationships with auxins in promoting plant cell division, we looked for Arabidopsis mutants with phenotypes which were affected by exogenously applied CKs. We have previously reported the isolation of the pasticcino mutants (pas1, pas2, and pas3) which are affected in both embryonic and vegetative development. Their phenotypes are similar to that of wild-type shoots which have been regenerated in vitro from explants, in the presence of an unbalanced auxin/CK ratio in the medium (12).The pas1-1 mutant was isolated from the transfer DNA (T-DNA) mutant collection of INRA-Centre de Versailles (2, 12). Here we describe the cloning of the PAS1 gene from the T-DNA-tagged pas1-1 allele. PAS1 codes for an immunophilin-like protein similar to the FK506-binding proteins (FKBP). We also demonstrate that the PAS1 mRNA steady-state level is increased in the presence of CK and that PAS1 gene expression is affected in the other pas mutants.     

Construction of a Plant Transient Expression Vector which Coexpresses the Marker β-glucuronidase

We have developed a plant transient expression vector that simultaneously expresses -glucuronidase from a Cauliflower mosaic virus promoter, and a test gene from a Figwort mosaic virus promoter. This vector, which is manipulated in E. coli, allows the testing of cell death inducing genes in plant cells. We have demonstrated the capability of this vector by expressing diphtheria toxin.     

miRPlant: an integrated tool for identification of plant miRNA from RNA sequencing data

Background

Small RNA sequencing is commonly used to identify novel miRNAs and to determine their expression levels in plants. There are several miRNA identification tools for animals such as miRDeep, miRDeep2 and miRDeep*. miRDeep-P was developed to identify plant miRNA using miRDeep’s probabilistic model of miRNA biogenesis, but it depends on several third party tools and lacks a user-friendly interface. The objective of our miRPlant program is to predict novel plant miRNA, while providing a user-friendly interface with improved accuracy of prediction.

Result

We have developed a user-friendly plant miRNA prediction tool called miRPlant. We show using 16 plant miRNA datasets from four different plant species that miRPlant has at least a 10% improvement in accuracy compared to miRDeep-P, which is the most popular plant miRNA prediction tool. Furthermore, miRPlant uses a Graphical User Interface for data input and output, and identified miRNA are shown with all RNAseq reads in a hairpin diagram.

Conclusions

We have developed miRPlant which extends miRDeep* to various plant species by adopting suitable strategies to identify hairpin excision regions and hairpin structure filtering for plants. miRPlant does not require any third party tools such as mapping or RNA secondary structure prediction tools. miRPlant is also the first plant miRNA prediction tool that dynamically plots miRNA hairpin structure with small reads for identified novel miRNAs. This feature will enable biologists to visualize novel pre-miRNA structure and the location of small RNA reads relative to the hairpin. Moreover, miRPlant can be easily used by biologists with limited bioinformatics skills.miRPlant and its manual are freely available at http://www.australianprostatecentre.org/research/software/mirplant or http://sourceforge.net/projects/mirplant/.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-275) contains supplementary material, which is available to authorized users.     

Understanding plant cell-wall remodelling during the symbiotic interaction between <Emphasis Type="Italic">Tuber melanosporum</Emphasis> and <Emphasis Type="Italic">Corylus avellana</Emphasis> using a carbohydrate microarray

Main conclusion

A combined approach, using a carbohydrate microarray as a support for genomic data, has revealed subtle plant cell-wall remodelling during Tuber melanosporum and Corylus avellana interaction. Cell walls are involved, to a great extent, in mediating plant–microbe interactions. An important feature of these interactions concerns changes in the cell-wall composition during interaction with other organisms. In ectomycorrhizae, plant and fungal cell walls come into direct contact, and represent the interface between the two partners. However, very little information is available on the re-arrangement that could occur within the plant and fungal cell walls during ectomycorrhizal symbiosis. Taking advantage of the Comprehensive Microarray Polymer Profiling (CoMPP) technology, the current study has had the aim of monitoring the changes that take place in the plant cell wall in Corylus avellana roots during colonization by the ascomycetous ectomycorrhizal fungus T. melanosporum. Additionally, genes encoding putative plant cell-wall degrading enzymes (PCWDEs) have been identified in the T. melanosporum genome, and RT-qPCRs have been performed to verify the expression of selected genes in fully developed C. avellana/T. melanosporum ectomycorrhizae. A localized degradation of pectin seems to occur during fungal colonization, in agreement with the growth of the ectomycorrhizal fungus through the middle lamella and with the fungal gene expression of genes acting on these polysaccharides.

     

Evolutionary Dynamics of the Cellulose Synthase Gene Superfamily in Grasses

Phylogenetic analyses of cellulose synthase (CesA) and cellulose synthase-like (Csl) families from the cellulose synthase gene superfamily were used to reconstruct their evolutionary origins and selection histories. Counterintuitively, genes encoding primary cell wall CesAs have undergone extensive expansion and diversification following an ancestral duplication from a secondary cell wall-associated CesA. Selection pressure across entire CesA and Csl clades appears to be low, but this conceals considerable variation within individual clades. Genes in the CslF clade are of particular interest because some mediate the synthesis of (1,3;1,4)-β-glucan, a polysaccharide characteristic of the evolutionarily successful grasses that is not widely distributed elsewhere in the plant kingdom. The phylogeny suggests that duplication of either CslF6 and/or CslF7 produced the ancestor of a highly conserved cluster of CslF genes that remain located in syntenic regions of all the grass genomes examined. A CslF6-specific insert encoding approximately 55 amino acid residues has subsequently been incorporated into the gene, or possibly lost from other CslFs, and the CslF7 clade has undergone a significant long-term shift in selection pressure. Homology modeling and molecular dynamics of the CslF6 protein were used to define the three-dimensional dispositions of individual amino acids that are subject to strong ongoing selection, together with the position of the conserved 55-amino acid insert that is known to influence the amounts and fine structures of (1,3;1,4)-β-glucans synthesized. These wall polysaccharides are attracting renewed interest because of their central roles as sources of dietary fiber in human health and for the generation of renewable liquid biofuels.Recent attempts to better understand the chemistry and biology of plant cell walls have been driven by the importance of these walls as biomass sources for biofuel production systems, as sources of dietary fiber that is increasingly recognized as being highly beneficial for human health, and as key components of livestock forage and fodder. Plant cell walls consist predominantly of polysaccharides and lignin. In addition to cellulose, walls contain a wide array of complex noncellulosic polysaccharides that vary across the plant kingdom (Carpita, 1996; Popper and Fry, 2003; Niklas, 2004; Popper and Tuohy, 2010). In the dicotyledons, pectic polysaccharides and xyloglucans predominate, although smaller amounts of heteroxylans and heteromannans are also found. In evolutionary terms, a major change in noncellulosic wall composition is observed with the emergence of the Poaceae family, which contains the grasses and important cereal species. In contrast to dicots, walls of the Poaceae have relatively low levels of pectic polysaccharides and xyloglucans and correspondingly higher levels of heteroxylans, which appear to constitute the core noncellulosic wall polysaccharides in this family. In addition, walls of the Poaceae often contain (1,3;1,4)-β-glucans, which are not widely distributed in dicotyledons or other monocotyledons (Carpita, 1996; Fincher, 2009).Following the identification of the genes that encode cellulose synthases, which were designated CesA genes (Pear et al., 1996; Arioli et al., 1998), analyses of EST databases quickly revealed that the CesA group of cellulose synthase genes was in fact just one clade of a much larger superfamily that contained up to about 50 genes in most land plants (Richmond and Somerville, 2000; Hazen et al., 2002). The other members of the large gene family were designated cellulose synthase-like genes (Csl), which represent several clades in the overall phylogeny of the superfamily (Supplemental Fig. S1). The plant CesA genes were shown to have both conserved and hypervariable regions (Delmer, 1999; Doblin et al., 2002) and, together with the related Csl genes, were predicted to be integral membrane proteins and to have conserved, active-site D,D,D,QxxRW amino acid sequences. The CesA and Csl genes are members of the GT2 family of glycosyltransferases (Cantarel et al., 2009; http://www.cazy.org/).Several of the Csl genes have now been implicated in the biosynthesis of noncellulosic wall polysaccharides. Certain CslA genes mediate mannan and glucomannan synthesis (Dhugga et al., 2004; Liepman et al., 2005). Genes in the CslC clade are believed to be involved in xyloglucan biosynthesis (Cocuron et al., 2007), while genes from both the CslF and CslH clades mediate (1,3;1,4)-β-glucan synthesis in the Poaceae (Burton et al., 2006; Doblin et al., 2009). The CslJ group of enzymes is also believed to be involved in (1,3;1,4)-β-glucan synthesis (Farrokhi et al., 2006; Fincher, 2009), but the phylogeny of this group of genes remains unresolved (Yin et al., 2009). The fact that the CslF group does not form a clade with the CslH and CslJ groups on the phylogenetic tree (Supplemental Fig. S1) led to the suggestion that the genes mediating (1,3;1,4)-β-glucan synthesis have evolved independently on more than one occasion (Doblin et al., 2009; Fincher, 2009).Against this background and considering the sequence similarities between genes in the cellulose synthase gene superfamily, we have used Bayesian phylogenetic analyses of these genes from seven fully sequenced taxa to reconstruct the evolutionary origins of the CesA and Csl families in the grasses and, in particular, to investigate the evolution of the CslF, CslH, and CslJ genes. The distributions of the genes across genomes were compared, CslF gene clusters were analyzed, and the rates of synonymous and nonsynonymous nucleotide substitution were estimated to assess and compare selection histories of individual members of clades within the gene superfamily. Finally, we have constructed a refined model of the barley CslF6 enzyme to observe how selection on specific residues and regions of the enzyme has operated in a structural and functional context.