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The selaginella genome identifies genetic changes associated with the evolution of vascular plants
(2011)
Vascular plants appeared similar to 410 million years ago, then diverged into several lineages of which only two survive: the euphyllophytes (ferns and seed plants) and the lycophytes. We report here the genome sequence of the lycophyte Selaginella moellendorffii (Selaginella), the first nonseed vascular plant genome reported. By comparing gene content in evolutionarily diverse taxa, we found that the transition from a gametophyte- to a sporophyte-dominated life cycle required far fewer new genes than the transition from a nonseed vascular to a flowering plant, whereas secondary metabolic genes expanded extensively and in parallel in the lycophyte and angiosperm lineages. Selaginella differs in posttranscriptional gene regulation, including small RNA regulation of repetitive elements, an absence of the trans-acting small interfering RNA pathway, and extensive RNA editing of organellar genes.
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 similar to 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.
Although the positive effect of elevated CO2 concentration [CO2] on plant growth is well known, it remains unclear whether global climate change will positively or negatively affect crop yields. In particular, relatively little is known about the role of hormone pathways in controlling the growth responses to elevated [CO2]. Here, we studied the impact of elevated [CO2] on plant biomass and metabolism in Arabidopsis (Arabidopsis thaliana) in relation to the availability of gibberellins (GAs). Inhibition of growth by the GA biosynthesis inhibitor paclobutrazol (PAC) at ambient [CO2] (350 mu mol CO2 mol(-1)) was reverted by elevated [CO2] (750 mu mol CO2 mol(-1)). Thus, we investigated the metabolic adjustment and modulation of gene expression in response to changes in growth of plants imposed by varying the GA regime in ambient and elevated [CO2]. In the presence of PAC (low-GA regime), the activities of enzymes involved in photosynthesis and inorganic nitrogen assimilation were markedly increased at elevated [CO2], whereas the activities of enzymes of organic acid metabolism were decreased. Under ambient [CO2], nitrate, amino acids, and protein accumulated upon PAC treatment; however, this was not the case when plants were grown at elevated [CO2]. These results suggest that only under ambient [CO2] is GA required for the integration of carbohydrate and nitrogen metabolism underlying optimal biomass determination. Our results have implications concerning the action of the Green Revolution genes in future environmental conditions.
Organisms have evolved endogenous biological clocks as internal timekeepers to coordinate metabolic processes with the external environment. Here, we seek to understand the mechanism of synchrony between the oscillator and products of metabolism known as Reactive Oxygen Species (ROS) in Arabidopsis thaliana. ROS-responsive genes exhibit a time-of-day-specific phase of expression under diurnal and circadian conditions, implying a role of the circadian clock in transcriptional regulation of these genes. Hydrogen peroxide production and scavenging also display time-of-day phases. Mutations in the core-clock regulator, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), affect the transcriptional regulation of ROS-responsive genes, ROS homeostasis, and tolerance to oxidative stress. Mis-expression of EARLY FLOWERING 3, LUX ARRHYTHMO, and TIMING OF CAB EXPRESSION 1 affect ROS production and transcription, indicating a global effect of the clock on the ROS network. We propose CCA1 as a master regulator of ROS homeostasis through association with the Evening Element in promoters of ROS genes in vivo to coordinate time-dependent responses to oxidative stress. We also find that ROS functions as an input signal that affects the transcriptional output of the clock, revealing an important link between ROS signaling and circadian output. Temporal coordination of ROS signaling by CCA1 and the reciprocal control of circadian output by ROS reveal a mechanistic link that allows plants to master oxidative stress responses.
Translatome and metabolome effects triggered by gibberellins during rosette growth in Arabidopsis
(2012)
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 (GA(3)) to monitor translational regulation of mRNAs globally. Gibberellin levels did not affect levels of carbohydrates in plants treated with PAC and/or GA(3). 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 GA(3) alone, or PAC plus GA(3). 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.
The assembly, sorting signals, and turnover of the tonoplast potassium channel AtTPK1 of Arabidopsis (Arabidopsis thaliana) were studied. We used transgenic Arabidopsis expressing a TPK1-green fluorescent protein (GFP) fusion or protoplasts transiently transformed with chimeric constructs based on domain exchange between TPK1 and TPK4, the only TPK family member not located at the tonoplast. The results show that TPK1-GFP is a dimer and that the newly synthesized polypeptides transiently interact with a thus-far unidentified 20-kD polypeptide. A subset of the TPK1-TPK4 chimeras were unable to assemble correctly and these remained located in the endoplasmic reticulum where they interacted with the binding protein chaperone. Therefore, TPK1 must assemble correctly to pass endoplasmic reticulum quality control. Substitution of the cytosolic C terminus of TPK4 with the corresponding domain of TPK1 was sufficient to allow tonoplast delivery, indicating that this domain contains tonoplast sorting information. Pulse-chase labeling indicated that TPK1-GFP has a half-life of at least 24 h. Turnover of the fusion protein involves internalization into the vacuole where the GFP domain is released. This indicates a possible mechanism for the turnover of tonoplast proteins.
Nuclear proteins play a central role in regulating gene expression. Their identification is important for understanding how the nuclear repertoire changes over time under different conditions. Nuclear proteins are often underrepresented in proteomic studies due to the frequently low abundance of proteins involved in regulatory processes. So far, only few studies describing the nuclear proteome of plant species have been published. Recently, the genome sequence of the unicellular green alga Chlamydomonas reinhardtii has been obtained and annotated, allowing the development of further detailed studies for this organism. However, a detailed description of its nuclear proteome has not been reported so far. Here, we present an analysis of the nuclear proteome of the sequenced Chlamydomonas strain cc503. Using LC-MS/MS, we identified 672 proteins from nuclei isolates with a maximum 1% peptide spectrum false discovery rate. Besides well-known proteins (e.g. histones), transcription factors and other transcriptional regulators (e.g. tubby and HMG) were identified. The presence of protein motifs in nuclear proteins was investigated by computational tools, and specific over-represented protein motifs were identified. This study provides new insights into the complexity of the nuclear environment and reveals novel putative protein targets for further studies of nuclear mechanisms.
With its homo-pentameric structure and calcium-dependent specificity for phosphocholine (PCh), human c-reactive protein (CRP) is produced by the liver and secreted in elevated quantities in response to inflammation. CRP is widely accepted as a cardiac marker, e.g. in point-of-care diagnostics, however, its heterologous expression has proven difficult. Here, we demonstrate the expression of CRP in different Escherichia coli strains as well as by in vitro transcription/translation. Although expression in these systems was straightforward, most of the protein that accumulated was insoluble. We therefore expanded our study to include the expression of CRP in two eukaryotic hosts, namely the yeast Kluyveromyces lactis and the protozoon Leishmania tarentolae. Both expression systems are optimized for secretion of recombinant proteins and here allowed successful expression of soluble CRP. We also demonstrate the purification of recombinant CRP from Leishmania growth medium; the purification of protein expressed from K. lactis was not successful. Functional and intact CRP pentamer is known to interact with PCh in Ca(2+)-dependent manner. In this report we verify the binding specificity of recombinant CRP from L tarentolae (2 mu g/mL culture medium) for PCh.
The balance between cellular proliferation and differentiation is a key aspect of development in multicellular organisms. Recent studies on Arabidopsis roots revealed distinct roles for different reactive oxygen species (ROS) in these processes. Modulation of the balance between ROS in proliferating cells and elongating cells is controlled at least in part at the transcriptional level. The effect of ROS on proliferation and differentiation is not specific for plants but appears to be conserved between prokaryotic and eukaryotic life forms. The ways in which ROS is received and how it affects cellular functioning is discussed from an evolutionary point of view. The different redox-sensing mechanisms that evolved ultimately result in the activation of gene regulatory networks that control cellular fate and decision-making. This review highlights the potential common origin of ROS sensing, indicating that organisms evolved similar strategies for utilizing ROS during development, and discusses ROS as an ancient universal developmental regulator.
Background: 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. Results: 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. Conclusions: 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.