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1.  Gene regulatory network inference using fused LASSO on multiple data sets 
Scientific Reports  2016;6:20533.
Devising computational methods to accurately reconstruct gene regulatory networks given gene expression data is key to systems biology applications. Here we propose a method for reconstructing gene regulatory networks by simultaneous consideration of data sets from different perturbation experiments and corresponding controls. The method imposes three biologically meaningful constraints: (1) expression levels of each gene should be explained by the expression levels of a small number of transcription factor coding genes, (2) networks inferred from different data sets should be similar with respect to the type and number of regulatory interactions, and (3) relationships between genes which exhibit similar differential behavior over the considered perturbations should be favored. We demonstrate that these constraints can be transformed in a fused LASSO formulation for the proposed method. The comparative analysis on transcriptomics time-series data from prokaryotic species, Escherichia coli and Mycobacterium tuberculosis, as well as a eukaryotic species, mouse, demonstrated that the proposed method has the advantages of the most recent approaches for regulatory network inference, while obtaining better performance and assigning higher scores to the true regulatory links. The study indicates that the combination of sparse regression techniques with other biologically meaningful constraints is a promising framework for gene regulatory network reconstructions.
PMCID: PMC4750075  PMID: 26864687
2.  ROS-mediated abiotic stress-induced programmed cell death in plants 
During the course of their ontogenesis plants are continuously exposed to a large variety of abiotic stress factors which can damage tissues and jeopardize the survival of the organism unless properly countered. While animals can simply escape and thus evade stressors, plants as sessile organisms have developed complex strategies to withstand them. When the intensity of a detrimental factor is high, one of the defense programs employed by plants is the induction of programmed cell death (PCD). This is an active, genetically controlled process which is initiated to isolate and remove damaged tissues thereby ensuring the survival of the organism. The mechanism of PCD induction usually includes an increase in the levels of reactive oxygen species (ROS) which are utilized as mediators of the stress signal. Abiotic stress-induced PCD is not only a process of fundamental biological importance, but also of considerable interest to agricultural practice as it has the potential to significantly influence crop yield. Therefore, numerous scientific enterprises have focused on elucidating the mechanisms leading to and controlling PCD in response to adverse conditions in plants. This knowledge may help develop novel strategies to obtain more resilient crop varieties with improved tolerance and enhanced productivity. The aim of the present review is to summarize the recent advances in research on ROS-induced PCD related to abiotic stress and the role of the organelles in the process.
PMCID: PMC4332301  PMID: 25741354
abiotic stress; programmed cell death; reactive oxygen species; signal transduction; stress adaptation
3.  PLAZA 3.0: an access point for plant comparative genomics 
Nucleic Acids Research  2014;43(Database issue):D974-D981.
Comparative sequence analysis has significantly altered our view on the complexity of genome organization and gene functions in different kingdoms. PLAZA 3.0 is designed to make comparative genomics data for plants available through a user-friendly web interface. Structural and functional annotation, gene families, protein domains, phylogenetic trees and detailed information about genome organization can easily be queried and visualized. Compared with the first version released in 2009, which featured nine organisms, the number of integrated genomes is more than four times higher, and now covers 37 plant species. The new species provide a wider phylogenetic range as well as a more in-depth sampling of specific clades, and genomes of additional crop species are present. The functional annotation has been expanded and now comprises data from Gene Ontology, MapMan, UniProtKB/Swiss-Prot, PlnTFDB and PlantTFDB. Furthermore, we improved the algorithms to transfer functional annotation from well-characterized plant genomes to other species. The additional data and new features make PLAZA 3.0 ( a versatile and comprehensible resource for users wanting to explore genome information to study different aspects of plant biology, both in model and non-model organisms.
PMCID: PMC4384038  PMID: 25324309
4.  The contribution of SERF1 to root-to-shoot signaling during salinity stress in rice 
Plant Signaling & Behavior  2014;9:e27540.
Stress perception and communication play important roles in the adaptation of plants to changing environmental conditions. Plant roots are the first organs to detect changes in the soil water potential induced by salt stress. In the presence of salinity stress, root-to-shoot communication occurs to adjust the growth of the whole plant. So far, the phytohormone abscisic acid (ABA), hydraulic signals and reactive oxygen species (ROS) have been proposed to mediate this communication under salt stress. Recently, we identified the rice transcription factor SALT-RESPONSIVE ERF1 (SERF1), which regulates a ROS-dependent transcriptional cascade in roots required for salinity tolerance. Upon salt stress, SERF1 knockout mutant plants show an increased leaf temperature as compared with wild type. As this occurs within the first 20 min of salt stress, we here evaluated the involvement of SERF1 in the perception of salt stress in the shoot. By metabolic profiling and expression analysis we show that the action of SERF1 in signal communication to the shoot is independent from ABA, but does affect the accumulation of ROS-related metabolites and transcripts under short-term salt stress.
PMCID: PMC4091250  PMID: 24451326
reactive oxygen species; transcription factor; metabolism; root-to-shoot; salt stress; Oryza sativa
5.  Reversal of senescence by N resupply to N-starved Arabidopsis thaliana: transcriptomic and metabolomic consequences 
Journal of Experimental Botany  2014;65(14):3975-3992.
Precocious senescence induced by nitrogen shortage can be reversed by N resupply. This study identifies the transcriptomic, metabolomic, and hormonal rearrangements underlying this process
Leaf senescence is a developmentally controlled process, which is additionally modulated by a number of adverse environmental conditions. Nitrogen shortage is a well-known trigger of precocious senescence in many plant species including crops, generally limiting biomass and seed yield. However, leaf senescence induced by nitrogen starvation may be reversed when nitrogen is resupplied at the onset of senescence. Here, the transcriptomic, hormonal, and global metabolic rearrangements occurring during nitrogen resupply-induced reversal of senescence in Arabidopsis thaliana were analysed. The changes induced by senescence were essentially in keeping with those previously described; however, these could, by and large, be reversed. The data thus indicate that plants undergoing senescence retain the capacity to sense and respond to the availability of nitrogen nutrition. The combined data are discussed in the context of the reversibility of the senescence programme and the evolutionary benefit afforded thereby. Future prospects for understanding and manipulating this process in both Arabidopsis and crop plants are postulated.
PMCID: PMC4106441  PMID: 24692653
Arabidopsis; gene expression; metabolomics; nitrogen limitation; senescence; transcriptome.
7.  Genome-Wide Identification of Regulatory Elements and Reconstruction of Gene Regulatory Networks of the Green Alga Chlamydomonas reinhardtii under Carbon Deprivation 
PLoS ONE  2013;8(11):e79909.
The unicellular green alga Chlamydomonas reinhardtii is a long-established model organism for studies on photosynthesis and carbon metabolism-related physiology. Under conditions of air-level carbon dioxide concentration [CO2], a carbon concentrating mechanism (CCM) is induced to facilitate cellular carbon uptake. CCM increases the availability of carbon dioxide at the site of cellular carbon fixation. To improve our understanding of the transcriptional control of the CCM, we employed FAIRE-seq (formaldehyde-assisted Isolation of Regulatory Elements, followed by deep sequencing) to determine nucleosome-depleted chromatin regions of algal cells subjected to carbon deprivation. Our FAIRE data recapitulated the positions of known regulatory elements in the promoter of the periplasmic carbonic anhydrase (Cah1) gene, which is upregulated during CCM induction, and revealed new candidate regulatory elements at a genome-wide scale. In addition, time series expression patterns of 130 transcription factor (TF) and transcription regulator (TR) genes were obtained for cells cultured under photoautotrophic condition and subjected to a shift from high to low [CO2]. Groups of co-expressed genes were identified and a putative directed gene-regulatory network underlying the CCM was reconstructed from the gene expression data using the recently developed IOTA (inner composition alignment) method. Among the candidate regulatory genes, two members of the MYB-related TF family, Lcr1 (Low-CO2 response regulator 1) and Lcr2 (Low-CO2 response regulator 2), may play an important role in down-regulating the expression of a particular set of TF and TR genes in response to low [CO2]. The results obtained provide new insights into the transcriptional control of the CCM and revealed more than 60 new candidate regulatory genes. Deep sequencing of nucleosome-depleted genomic regions indicated the presence of new, previously unknown regulatory elements in the C. reinhardtii genome. Our work can serve as a basis for future functional studies of transcriptional regulator genes and genomic regulatory elements in Chlamydomonas.
PMCID: PMC3816576  PMID: 24224019
8.  Could ROS signals drive tissue-specific clocks? 
Transcription  2013;4(5):206-208.
Circadian clocks have emerged to tune the physiology of organisms to periodic changes in the environment in a dynamic fashion. Negative implications of circadian disruptions in humans, animals and plants have encouraged extensive studies of clock-controlled biological processes in various model species. Recently, it has been shown that the transcription-dependent and -independent biological oscillators are largely driven by cellular oxidative cycles that are intrinsically linked with metabolism. Essentially, the clock is viewed as an integrated network that encompasses cytosolic, genetic and metabolic dimensions. Furthermore, in multicellular organisms, the clock network is organized in a tissue-specific manner. Here we discuss questions that remain unanswered: How do these dimensions communicate with each other and how do tissue-specific clocks exchange temporal information within multicellular organisms?
PMCID: PMC4114656  PMID: 24135705
CIRCADIAN CLOCK ASSOCIATED 1; Reactive Oxygen Species; circadian outputs; diurnal; metabolic timer; metabolism; tissue-specific
9.  Promotion of growth by elevated carbon dioxide is coordinated through a flexible transcriptional network in Arabidopsis 
Plant Signaling & Behavior  2013;8(3):e23356.
Although gibberellins (GAs) promote many developmental responses in plants, little is known about how the hormone interacts with environmental signals at the molecular level for regulating plant growth. Recently, we have demonstrated that inhibition of growth by the GA biosynthesis inhibitor paclobutrazol (PAC) at ambient [CO2] (350 µmol CO2 mol-1) is reverted by elevated [CO2] (750 μmol CO2 mol-1). Our finding points to an important role of elevated [CO2] as a signal allowing higher growth rates of low-GA plants. GA promotes plant growth via a complex transcriptional network that integrates multiple signaling pathways. Herein, we discuss how elevated [CO2] stimulates biomass accumulation in a GA-independent manner by regulating the expression of growth-related genes.
PMCID: PMC3676502  PMID: 23333969
elevated carbon dioxide concentration; transcription factor; gibberellin; growth; paclobutrazol
10.  Extensive Modulation of the Transcription Factor Transcriptome during Somatic Embryogenesis in Arabidopsis thaliana 
PLoS ONE  2013;8(7):e69261.
Molecular mechanisms controlling plant totipotency are largely unknown and studies on somatic embryogenesis (SE), the process through which already differentiated cells reverse their developmental program and become embryogenic, provide a unique means for deciphering molecular mechanisms controlling developmental plasticity of somatic cells. Among various factors essential for embryogenic transition of somatic cells transcription factors (TFs), crucial regulators of genetic programs, are believed to play a central role. Herein, we used quantitative real-time polymerase chain reaction (qRT-PCR) to identify TF genes affected during SE induced by in vitro culture in Arabidopsis thaliana. Expression profiles of 1,880 TFs were evaluated in the highly embryogenic Col-0 accession and the non-embryogenic tanmei/emb2757 mutant. Our study revealed 729 TFs whose expression changes during the 10-days incubation period of SE; 141 TFs displayed distinct differences in expression patterns in embryogenic versus non-embryogenic cultures. The embryo-induction stage of SE occurring during the first 5 days of culture was associated with a robust and dramatic change of the TF transcriptome characterized by the drastic up-regulation of the expression of a great majority (over 80%) of the TFs active during embryogenic culture. In contrast to SE induction, the advanced stage of embryo formation showed attenuation and stabilization of transcript levels of many TFs. In total, 519 of the SE-modulated TFs were functionally annotated and transcripts related with plant development, phytohormones and stress responses were found to be most abundant. The involvement of selected TFs in SE was verified using T-DNA insertion lines and a significantly reduced embryogenic response was found for the majority of them. This study provides comprehensive data focused on the expression of TF genes during SE and suggests directions for further research on functional genomics of SE.
PMCID: PMC3714258  PMID: 23874927
11.  Arabidopsis NAC transcription factor JUNGBRUNNEN1 affects thermomemory-associated genes and enhances heat stress tolerance in primed and unprimed conditions 
Plant Signaling & Behavior  2012;7(12):1518-1521.
We recently reported that the NAC transcription factor JUNGBRUNNEN1 (JUB1; ANAC042) extends longevity and increases tolerance to heat stress in Arabidopsis thaliana when overexpressed, while the opposite is observed in jub1-1 knock-down lines. Here we extend our previous findings by demonstrating that JUB1 also positively regulates plant survival under heat stress when plants were treated by a prior moderate (and non-lethal) temperature regime (so-called priming). We further find that JUB1 shows thermomemory-related expression, similar to two other genes previously reported to be important for thermopriming, i.e., HSFA2, encoding a heat shock factor, and HSA32, encoding a heat shock protein. Our analysis also identifies ASCORBATE PEROXIDASE2 (APX2) and the heat shock protein genes HSP18.2 and HSP21 as thermomemory-expressed genes, revealing them as new candidates for studies to decode the molecular processes controlling thermopriming.
PMCID: PMC3578882  PMID: 23073024
Abiotic stress; ANAC042; heat stress tolerance; hydrogen peroxide; JUB1; molecular memory; reactive oxygen species; thermopriming
12.  Network-Based Segmentation of Biological Multivariate Time Series 
PLoS ONE  2013;8(5):e62974.
Molecular phenotyping technologies (e.g., transcriptomics, proteomics, and metabolomics) offer the possibility to simultaneously obtain multivariate time series (MTS) data from different levels of information processing and metabolic conversions in biological systems. As a result, MTS data capture the dynamics of biochemical processes and components whose couplings may involve different scales and exhibit temporal changes. Therefore, it is important to develop methods for determining the time segments in MTS data, which may correspond to critical biochemical events reflected in the coupling of the system’s components. Here we provide a novel network-based formalization of the MTS segmentation problem based on temporal dependencies and the covariance structure of the data. We demonstrate that the problem of partitioning MTS data into segments to maximize a distance function, operating on polynomially computable network properties, often used in analysis of biological network, can be efficiently solved. To enable biological interpretation, we also propose a breakpoint-penalty (BP-penalty) formulation for determining MTS segmentation which combines a distance function with the number/length of segments. Our empirical analyses of synthetic benchmark data as well as time-resolved transcriptomics data from the metabolic and cell cycles of Saccharomyces cerevisiae demonstrate that the proposed method accurately infers the phases in the temporal compartmentalization of biological processes. In addition, through comparison on the same data sets, we show that the results from the proposed formalization of the MTS segmentation problem match biological knowledge and provide more rigorous statistical support in comparison to the contending state-of-the-art methods.
PMCID: PMC3646968  PMID: 23667552
13.  Effect of salt stress on genes encoding translation-associated proteins in Arabidopsis thaliana 
Plant Signaling & Behavior  2012;7(9):1095-1102.
Salinity negatively affects plant growth and disturbs chloroplast integrity. Here, we aimed at identifying salt-responsive translation-related genes in Arabidopsis thaliana with an emphasis on those encoding plastid-located proteins. We used quantitative real-time PCR to test the expression of 170 genes after short-term salt stress (up to 24 h) and identified several genes affected by the stress including: PRPL11, encoding plastid ribosomal protein L11, ATAB2, encoding a chloroplast-located RNA-binding protein presumably functioning as an activator of translation, and PDF1B, encoding a peptide deformylase involved in N-formyl group removal from nascent proteins synthesized in chloroplasts. These genes were previously shown to have important functions in chloroplast biology and may therefore represent new targets for biotechnological optimization of salinity tolerance.
PMCID: PMC3489636  PMID: 22899071
Arabidopsis thaliana; gene expression; salt stress; ribosomal protein; translation; variation of information; clustering; dynamic time warping
14.  Translatome and metabolome effects triggered by gibberellins during rosette growth in Arabidopsis 
Journal of Experimental Botany  2012;63(7):2769-2786.
Although gibberellins (GAs) are well known for their growth control function, little is known about their effects on primary metabolism. Here the modulation of gene expression and metabolic adjustment in response to changes in plant (Arabidopsis thaliana) growth imposed on varying the gibberellin regime were evaluated. Polysomal mRNA populations were profiled following treatment of plants with paclobutrazol (PAC), an inhibitor of GA biosynthesis, and gibberellic acid (GA3) to monitor translational regulation of mRNAs globally. Gibberellin levels did not affect levels of carbohydrates in plants treated with PAC and/or GA3. However, the tricarboxylic acid cycle intermediates malate and fumarate, two alternative carbon storage molecules, accumulated upon PAC treatment. Moreover, an increase in nitrate and in the levels of the amino acids was observed in plants grown under a low GA regime. Only minor changes in amino acid levels were detected in plants treated with GA3 alone, or PAC plus GA3. Comparison of the molecular changes at the transcript and metabolite levels demonstrated that a low GA level mainly affects growth by uncoupling growth from carbon availability. These observations, together with the translatome changes, reveal an interaction between energy metabolism and GA-mediated control of growth to coordinate cell wall extension, secondary metabolism, and lipid metabolism.
PMCID: PMC3346235  PMID: 22291129
Gibberellin; growth; paclobutrazol; primary metabolism; translatome
15.  Transcription factor OsHsfC1b regulates salt tolerance and development in Oryza sativa ssp. japonica 
AoB Plants  2012;2012:pls011.
The paper describes the functional analysis of a class C heat shock transcription factor from rice (Oryza sativa). OsHsfC1b is shown to play a role in ABA-mediated salt stress tolerance and is required for plant growth under non-stress conditions.
Background and aims
Salt stress leads to attenuated growth and productivity in rice. Transcription factors like heat shock factors (HSFs) represent central regulators of stress adaptation. Heat shock factors of the classes A and B are well established as regulators of thermal and non-thermal stress responses in plants; however, the role of class C HSFs is unknown. Here we characterized the function of the OsHsfC1b (Os01g53220) transcription factor from rice.
We analysed the expression of OsHsfC1b in the rice japonica cultivars Dongjin and Nipponbare exposed to salt stress as well as after mannitol, abscisic acid (ABA) and H2O2 treatment. For functional characterization of OsHsfC1b, we analysed the physiological response of a T-DNA insertion line (hsfc1b) and two artificial micro-RNA (amiRNA) knock-down lines to salt, mannitol and ABA treatment. In addition, we quantified the expression of small Heat Shock Protein (sHSP) genes and those related to signalling and ion homeostasis by quantitative real-time polymerase chain reaction in roots exposed to salt. The subcellular localization of OsHsfC1b protein fused to green fluorescent protein (GFP) was determined in Arabidopsis mesophyll cell protoplasts.
Principal results
Expression of OsHsfC1b was induced by salt, mannitol and ABA, but not by H2O2. Impaired function of OsHsfC1b in the hsfc1b mutant and the amiRNA lines led to decreased salt and osmotic stress tolerance, increased sensitivity to ABA, and temporal misregulation of salt-responsive genes involved in signalling and ion homeostasis. Furthermore, sHSP genes showed enhanced expression in knock-down plants under salt stress. We observed retarded growth of hsfc1b and knock-down lines in comparison with control plants under non-stress conditions. Transient expression of OsHsfC1b fused to GFP in protoplasts revealed nuclear localization of the transcription factor.
OsHsfC1b plays a role in ABA-mediated salt stress tolerance in rice. Furthermore, OsHsfC1b is involved in the response to osmotic stress and is required for plant growth under non-stress conditions.
PMCID: PMC3357053  PMID: 22616023
16.  Expression of ROS-responsive genes and transcription factors after metabolic formation of H2O2 in chloroplasts 
Glycolate oxidase (GO) catalyses the oxidation of glycolate to glyoxylate, thereby consuming O2 and producing H2O2. In this work, Arabidopsis thaliana plants expressing GO in the chloroplasts (GO plants) were used to assess the expressional behavior of reactive oxygen species (ROS)-responsive genes and transcription factors (TFs) after metabolic induction of H2O2 formation in chloroplasts. In this organelle, GO uses the glycolate derived from the oxygenase activity of RubisCO. Here, to identify genes responding to an abrupt production of H2O2 in chloroplasts we used quantitative real-time PCR (qRT-PCR) to test the expression of 187 ROS-responsive genes and 1880 TFs after transferring GO and wild-type (WT) plants grown at high CO2 levels to ambient CO2 concentration. Our data revealed coordinated expression changes of genes of specific functional networks 0.5 h after metabolic induction of H2O2 production in GO plants, including the induction of indole glucosinolate and camalexin biosynthesis genes. Comparative analysis using available microarray data suggests that signals for the induction of these genes through H2O2 may originate in the chloroplast. The TF profiling indicated an up-regulation in GO plants of a group of genes involved in the regulation of proanthocyanidin and anthocyanin biosynthesis. Moreover, the upregulation of expression of TF and TF-interacting proteins affecting development (e.g., cell division, stem branching, flowering time, flower development) would impact growth and reproductive capacity, resulting in altered development under conditions that promote the formation of H2O2.
PMCID: PMC3485569  PMID: 23125844
glycolate oxidase; H2O2; ROS-responsive genes; transcription factors
17.  The compact Selaginella genome identifies changes in gene content associated with the evolution of vascular plants 
Banks, Jo Ann | Nishiyama, Tomoaki | Hasebe, Mitsuyasu | Bowman, John L. | Gribskov, Michael | dePamphilis, Claude | Albert, Victor A. | Aono, Naoki | Aoyama, Tsuyoshi | Ambrose, Barbara A. | Ashton, Neil W. | Axtell, Michael J. | Barker, Elizabeth | Barker, Michael S. | Bennetzen, Jeffrey L. | Bonawitz, Nicholas D. | Chapple, Clint | Cheng, Chaoyang | Correa, Luiz Gustavo Guedes | Dacre, Michael | DeBarry, Jeremy | Dreyer, Ingo | Elias, Marek | Engstrom, Eric M. | Estelle, Mark | Feng, Liang | Finet, Cédric | Floyd, Sandra K. | Frommer, Wolf B. | Fujita, Tomomichi | Gramzow, Lydia | Gutensohn, Michael | Harholt, Jesper | Hattori, Mitsuru | Heyl, Alexander | Hirai, Tadayoshi | Hiwatashi, Yuji | Ishikawa, Masaki | Iwata, Mineko | Karol, Kenneth G. | Koehler, Barbara | Kolukisaoglu, Uener | Kubo, Minoru | Kurata, Tetsuya | Lalonde, Sylvie | Li, Kejie | Li, Ying | Litt, Amy | Lyons, Eric | Manning, Gerard | Maruyama, Takeshi | Michael, Todd P. | Mikami, Koji | Miyazaki, Saori | Morinaga, Shin-ichi | Murata, Takashi | Mueller-Roeber, Bernd | Nelson, David R. | Obara, Mari | Oguri, Yasuko | Olmstead, Richard G. | Onodera, Naoko | Petersen, Bent Larsen | Pils, Birgit | Prigge, Michael | Rensing, Stefan A. | Riaño-Pachón, Diego Mauricio | Roberts, Alison W. | Sato, Yoshikatsu | Scheller, Henrik Vibe | Schulz, Burkhard | Schulz, Christian | Shakirov, Eugene V. | Shibagaki, Nakako | Shinohara, Naoki | Shippen, Dorothy E. | Sørensen, Iben | Sotooka, Ryo | Sugimoto, Nagisa | Sugita, Mamoru | Sumikawa, Naomi | Tanurdzic, Milos | Theißen, Günter | Ulvskov, Peter | Wakazuki, Sachiko | Weng, Jing-Ke | Willats, William W.G.T. | Wipf, Daniel | Wolf, Paul G. | Yang, Lixing | Zimmer, Andreas D. | Zhu, Qihui | Mitros, Therese | Hellsten, Uffe | Loqué, Dominique | Otillar, Robert | Salamov, Asaf | Schmutz, Jeremy | Shapiro, Harris | Lindquist, Erika | Lucas, Susan | Rokhsar, Daniel | Grigoriev, Igor V.
Science (New York, N.Y.)  2011;332(6032):960-963.
Vascular plants appeared ~410 million years ago then diverged into several lineages of which only two survive: the euphyllophytes (ferns and seed plants) and the lycophytes (1). We report here the genome sequence of the lycophyte Selaginella moellendorffii (Selaginella), the first non-seed vascular plant genome reported. By comparing gene content in evolutionary diverse taxa, we found that the transition from a gametophyte- to sporophyte-dominated life cycle required far fewer new genes than the transition from a non-seed vascular to a flowering plant, while secondary metabolic genes expanded extensively and in parallel in the lycophyte and angiosperm lineages. Selaginella differs in post-transcriptional gene regulation, including small RNA regulation of repetitive elements, an absence of the tasiRNA pathway and extensive RNA editing of organellar genes.
PMCID: PMC3166216  PMID: 21551031
18.  Positional Information Resolves Structural Variations and Uncovers an Evolutionarily Divergent Genetic Locus in Accessions of Arabidopsis thaliana 
Genome sequencing of closely related individuals has yielded valuable insights that link genome evolution to phenotypic variations. However, advancement in sequencing technology has also led to an escalation in the number of poor quality–drafted genomes assembled based on reference genomes that can have highly divergent or haplotypic regions. The self-fertilizing nature of Arabidopsis thaliana poses an advantage to sequencing projects because its genome is mostly homozygous. To determine the accuracy of an Arabidopsis drafted genome in less conserved regions, we performed a resequencing experiment on a ∼371-kb genomic interval in the Landsberg erecta (Ler-0) accession. We identified novel structural variations (SVs) between Ler-0 and the reference accession Col-0 using a long-range polymerase chain reaction approach to generate an Illumina data set that has positional information, that is, a data set with reads that map to a known location. Positional information is important for accurate genome assembly and the resolution of SVs particularly in highly duplicated or repetitive regions. Sixty-one regions with misassembly signatures were identified from the Ler-0 draft, suggesting the presence of novel SVs that are not represented in the draft sequence. Sixty of those were resolved by iterative mapping using our data set. Fifteen large indels (>100 bp) identified from this study were found to be located either within protein-coding regions or upstream regulatory regions, suggesting the formation of novel alleles or altered regulation of existing genes in Ler-0. We propose future genome-sequencing experiments to follow a clone-based approach that incorporates positional information to ultimately reveal haplotype-specific differences between accessions.
PMCID: PMC3157834  PMID: 21622917
haplotype; allelic variants; drafted genomes; genome partitioning; comparative genomics
19.  High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection 
PLoS ONE  2011;6(4):e18900.
Protein expression in heterologous hosts for functional studies is a cumbersome effort. Here, we report a superior platform for parallel protein expression in vivo and in vitro. The platform combines highly efficient ligation-independent cloning (LIC) with instantaneous detection of expressed proteins through N- or C-terminal fusions to infrared fluorescent protein (IFP). For each open reading frame, only two PCR fragments are generated (with three PCR primers) and inserted by LIC into ten expression vectors suitable for protein expression in microbial hosts, including Escherichia coli, Kluyveromyces lactis, Pichia pastoris, the protozoon Leishmania tarentolae, and an in vitro transcription/translation system. Accumulation of IFP-fusion proteins is detected by infrared imaging of living cells or crude protein extracts directly after SDS-PAGE without additional processing. We successfully employed the LIC-IFP platform for in vivo and in vitro expression of ten plant and fungal proteins, including transcription factors and enzymes. Using the IFP reporter, we additionally established facile methods for the visualisation of protein-protein interactions and the detection of DNA-transcription factor interactions in microtiter and gel-free format. We conclude that IFP represents an excellent reporter for high-throughput protein expression and analysis, which can be easily extended to numerous other expression hosts using the setup reported here.
PMCID: PMC3082538  PMID: 21541323
20.  ORS1, an H2O2-Responsive NAC Transcription Factor, Controls Senescence in Arabidopsis thaliana 
Molecular Plant  2011;4(2):346-360.
We report here that ORS1, a previously uncharacterized member of the NAC transcription factor family, controls leaf senescence in Arabidopsis thaliana. Overexpression of ORS1 accelerates senescence in transgenic plants, whereas its inhibition delays it. Genes acting downstream of ORS1 were identified by global expression analysis using transgenic plants producing dexamethasone-inducible ORS1–GR fusion protein. Of the 42 up-regulated genes, 30 (∼70%) were previously shown to be up-regulated during age-dependent senescence. We also observed that 32 (∼76%) of the ORS1-dependent genes were induced by long-term (4 d), but not short-term (6 h) salinity stress (150 mM NaCl). Furthermore, expression of 16 and 24 genes, respectively, was induced after 1 and 5 h of treatment with hydrogen peroxide (H2O2), a reactive oxygen species known to accumulate during salinity stress. ORS1 itself was found to be rapidly and strongly induced by H2O2 treatment in both leaves and roots. Using in vitro binding site selection, we determined the preferred binding motif of ORS1 and found it to be present in half of the ORS1-dependent genes. ORS1 is a paralog of ORE1/ANAC092/AtNAC2, a previously reported regulator of leaf senescence. Phylogenetic footprinting revealed evolutionary conservation of the ORS1 and ORE1 promoter sequences in different Brassicaceae species, indicating strong positive selection acting on both genes. We conclude that ORS1, similarly to ORE1, triggers expression of senescence-associated genes through a regulatory network that may involve cross-talk with salt- and H2O2-dependent signaling pathways.
PMCID: PMC3063519  PMID: 21303842
NAC transcription factor; leaf senescence; gene expression; gene regulatory network; hydrogen peroxide
21.  Different sequence signatures in the upstream regions of plant and animal tRNA genes shape distinct modes of regulation 
Nucleic Acids Research  2010;39(8):3331-3339.
In eukaryotes, the transcription of tRNA genes is initiated by the concerted action of transcription factors IIIC (TFIIIC) and IIIB (TFIIIB) which direct the recruitment of polymerase III. While TFIIIC recognizes highly conserved, intragenic promoter elements, TFIIIB binds to the non-coding 5′-upstream regions of the tRNA genes. Using a systematic bioinformatic analysis of 11 multicellular eukaryotic genomes we identified a highly conserved TATA motif followed by a CAA-motif in the tRNA upstream regions of all plant genomes. Strikingly, the 5′-flanking tRNA regions of the animal genomes are highly heterogeneous and lack a common conserved sequence signature. Interestingly, in the animal genomes the tRNA species that read the same codon share conserved motifs in their upstream regions. Deep-sequencing analysis of 16 human tissues revealed multiple splicing variants of two of the TFIIIB subunits, Bdp1 and Brf1, with tissue-specific expression patterns. These multiple forms most likely modulate the TFIIIB–DNA interactions and explain the lack of a uniform signature motif in the tRNA upstream regions of animal genomes. The anticodon-dependent 5′-flanking motifs provide a possible mechanism for independent regulation of the tRNA transcription in various human tissues.
PMCID: PMC3082873  PMID: 21138970
22.  Salt-triggered expression of the ANAC092-dependent senescence regulon in Arabidopsis thaliana 
Plant Signaling & Behavior  2010;5(6):733-735.
The NAC domain transcription factor ANAC092 plays a central role in leaf senescence in Arabidopsis thaliana. We recently identified 170 genes whose expression increases upon activation of ANAC092 in a chemically (estradiol) controlled experimental set-up, 78 of which are known senescence-associated genes (SAGs). In accordance with the well-known phenomenon that salt stress promotes early leaf senescence in many plant species, we previously observed salt stress-enhanced expression of many SAGs of the ANAC092 regulon. Global expression profiling now revealed that 36 genes, representing 46% of all ANAC092 downstream SAGs, are induced by long-term (4 days) salt stress in shoots of Arabidopsis, whereas short-term stress (6 hours) only slightly affects gene expression. Expression analysis also showed that 14 of the 36 genes are induced by hydrogen peroxide (H2O2) treatment. Additionally, 15 senescence-associated NAC genes (senNACs), including ANAC092, respond to H2O2 exposure. Our data support the model that salt-triggered senescence is at least partly mediated through the ANAC092 gene regulatory network. Other senNACs most likely contribute to the coordination of this process, potentially in concert with H2O2-mediated signaling.
PMCID: PMC3001574  PMID: 20404534
ANAC092; binding site; hydrogen peroxide; longevity; NAC transcription factor; ORE1; reactive oxygen species; regulon; salt stress; senescence
23.  Genome-Wide Phylogenetic Comparative Analysis of Plant Transcriptional Regulation: A Timeline of Loss, Gain, Expansion, and Correlation with Complexity 
Evolutionary retention of duplicated genes encoding transcription-associated proteins (TAPs, comprising transcription factors and other transcriptional regulators) has been hypothesized to be positively correlated with increasing morphological complexity and paleopolyploidizations, especially within the plant kingdom. Here, we present the most comprehensive set of classification rules for TAPs and its application for genome-wide analyses of plants and algae. Using a dated species tree and phylogenetic comparative (PC) analyses, we define the timeline of TAP loss, gain, and expansion among Viridiplantae and find that two major bursts of gain/expansion occurred, coinciding with the water-to-land transition and the radiation of flowering plants. For the first time, we provide PC proof for the long-standing hypothesis that TAPs are major driving forces behind the evolution of morphological complexity, the latter in Plantae being shaped significantly by polyploidization and subsequent biased paleolog retention. Principal component analysis incorporating the number of TAPs per genome provides an alternate and significant proxy for complexity, ideally suited for PC genomics. Our work lays the ground for further interrogation of the shaping of gene regulatory networks underlying the evolution of organism complexity.
PMCID: PMC2997552  PMID: 20644220
transcription factor; evolution; Plantae; phylogenetic comparative methods; morphological complexity
24.  The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions 
Merchant, Sabeeha S. | Prochnik, Simon E. | Vallon, Olivier | Harris, Elizabeth H. | Karpowicz, Steven J. | Witman, George B. | Terry, Astrid | Salamov, Asaf | Fritz-Laylin, Lillian K. | Maréchal-Drouard, Laurence | Marshall, Wallace F. | Qu, Liang-Hu | Nelson, David R. | Sanderfoot, Anton A. | Spalding, Martin H. | Kapitonov, Vladimir V. | Ren, Qinghu | Ferris, Patrick | Lindquist, Erika | Shapiro, Harris | Lucas, Susan M. | Grimwood, Jane | Schmutz, Jeremy | Cardol, Pierre | Cerutti, Heriberto | Chanfreau, Guillaume | Chen, Chun-Long | Cognat, Valérie | Croft, Martin T. | Dent, Rachel | Dutcher, Susan | Fernández, Emilio | Ferris, Patrick | Fukuzawa, Hideya | González-Ballester, David | González-Halphen, Diego | Hallmann, Armin | Hanikenne, Marc | Hippler, Michael | Inwood, William | Jabbari, Kamel | Kalanon, Ming | Kuras, Richard | Lefebvre, Paul A. | Lemaire, Stéphane D. | Lobanov, Alexey V. | Lohr, Martin | Manuell, Andrea | Meier, Iris | Mets, Laurens | Mittag, Maria | Mittelmeier, Telsa | Moroney, James V. | Moseley, Jeffrey | Napoli, Carolyn | Nedelcu, Aurora M. | Niyogi, Krishna | Novoselov, Sergey V. | Paulsen, Ian T. | Pazour, Greg | Purton, Saul | Ral, Jean-Philippe | Riaño-Pachón, Diego Mauricio | Riekhof, Wayne | Rymarquis, Linda | Schroda, Michael | Stern, David | Umen, James | Willows, Robert | Wilson, Nedra | Zimmer, Sara Lana | Allmer, Jens | Balk, Janneke | Bisova, Katerina | Chen, Chong-Jian | Elias, Marek | Gendler, Karla | Hauser, Charles | Lamb, Mary Rose | Ledford, Heidi | Long, Joanne C. | Minagawa, Jun | Page, M. Dudley | Pan, Junmin | Pootakham, Wirulda | Roje, Sanja | Rose, Annkatrin | Stahlberg, Eric | Terauchi, Aimee M. | Yang, Pinfen | Ball, Steven | Bowler, Chris | Dieckmann, Carol L. | Gladyshev, Vadim N. | Green, Pamela | Jorgensen, Richard | Mayfield, Stephen | Mueller-Roeber, Bernd | Rajamani, Sathish | Sayre, Richard T. | Brokstein, Peter | Dubchak, Inna | Goodstein, David | Hornick, Leila | Huang, Y. Wayne | Jhaveri, Jinal | Luo, Yigong | Martínez, Diego | Ngau, Wing Chi Abby | Otillar, Bobby | Poliakov, Alexander | Porter, Aaron | Szajkowski, Lukasz | Werner, Gregory | Zhou, Kemin | Grigoriev, Igor V. | Rokhsar, Daniel S. | Grossman, Arthur R.
Science (New York, N.Y.)  2007;318(5848):245-250.
Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the ∼120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.
PMCID: PMC2875087  PMID: 17932292
25.  A highly efficient pipeline for protein expression in Leishmania tarentolae using infrared fluorescence protein as marker 
Leishmania tarentolae, a unicellular eukaryotic protozoan, has been established as a novel host for recombinant protein production in recent years. Current protocols for protein expression in Leishmania are, however, time consuming and require extensive lab work in order to identify well-expressing cell lines. Here we established an alternative protein expression work-flow that employs recently engineered infrared fluorescence protein (IFP) as a suitable and easy-to-handle reporter protein for recombinant protein expression in Leishmania. As model proteins we tested three proteins from the plant Arabidopsis thaliana, including a NAC and a type-B ARR transcription factor.
IFP and IFP fusion proteins were expressed in Leishmania and rapidly detected in cells by deconvolution microscopy and in culture by infrared imaging of 96-well microtiter plates using small cell culture volumes (2 μL - 100 μL). Motility, shape and growth of Leishmania cells were not impaired by intracellular accumulation of IFP. In-cell detection of IFP and IFP fusion proteins was straightforward already at the beginning of the expression pipeline and thus allowed early pre-selection of well-expressing Leishmania clones. Furthermore, IFP fusion proteins retained infrared fluorescence after electrophoresis in denaturing SDS-polyacrylamide gels, allowing direct in-gel detection without the need to disassemble cast protein gels. Thus, parameters for scaling up protein production and streamlining purification routes can be easily optimized when employing IFP as reporter.
Using IFP as biosensor we devised a protocol for rapid and convenient protein expression in Leishmania tarentolae. Our expression pipeline is superior to previously established methods in that it significantly reduces the hands-on-time and work load required for identifying well-expressing clones, refining protein production parameters and establishing purification protocols. The facile in-cell and in-gel detection tools built on IFP make Leishmania amenable for high-throughput expression of proteins from plant and animal sources.
PMCID: PMC2874516  PMID: 20459748

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