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A large-scale metabolic quantitative trait loci (mQTL) analysis was performed on the well-characterized Solanum pennellii introgression lines to investigate the genomic regions associated with secondary metabolism in tomato fruit pericarp. In total, 679 mQTLs were detected across the 76 introgression lines. Heritability analyses revealed that mQTLs of secondary metabolism were less affected by environment than mQTLs of primary metabolism. Network analysis allowed us to assess the interconnectivity of primary and secondary metabolism as well as to compare and contrast their respective associations with morphological traits. Additionally, we applied a recently established real-time quantitative PCR platform to gain insight into transcriptional control mechanisms of a subset of the mQTLs, including those for hydroxycinnamates, acyl-sugar, naringenin chalcone, and a range of glycoalkaloids. Intriguingly, many of these compounds displayed a dominant-negative mode of inheritance, which is contrary to the conventional wisdom that secondary metabolite contents decreased on domestication. We additionally performed an exemplary evaluation of two candidate genes for glycolalkaloid mQTLs via the use of virus-induced gene silencing. The combined data of this study were compared with previous results on primary metabolism obtained from the same material and to other studies of natural variance of secondary metabolism.
To gain a deeper understanding of the mechanisms behind biomass accumulation, it is important to study plant growth behavior. Manually phenotyping large sets of plants requires important human resources and expertise and is typically not feasible for detection of weak growth phenotypes. Here, we established an automated growth phenotyping pipeline for Arabidopsis thaliana to aid researchers in comparing growth behaviors of different genotypes.
The analysis pipeline includes automated image analysis of two-dimensional digital plant images and evaluation of manually annotated information of growth stages. It employs linear mixed-effects models to quantify genotype effects on total rosette area and relative leaf growth rate (RLGR) and ANOVAs to quantify effects on developmental times.
Using the system, a single researcher can phenotype up to 7000 plants d(-1). Technical variance is very low (typically < 2%). We show quantitative results for the growth-impaired starch-excessmutant sex4-3 and the growth-enhancedmutant grf9.
We show that recordings of environmental and developmental variables reduce noise levels in the phenotyping datasets significantly and that careful examination of predictor variables (such as d after sowing or germination) is crucial to avoid exaggerations of recorded phenotypes and thus biased conclusions.
Glycolate oxidase (GO) catalyses the oxidation of glycolate to glyoxylate, thereby consuming O-2 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 IF and IF 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.
ORS1, an H2O2-Responsive NAC Transcription Factor, Controls Senescence in Arabidopsis thaliana
(2011)
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 (similar to 70%) were previously shown to be up-regulated during age-dependent senescence. We also observed that 32 (similar to 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.
A balance to death
(2018)
Leaf senescence plays a crucial role in nutrient recovery in late-stage plant development and requires vast transcriptional reprogramming by transcription factors such as ORESARA1 (ORE1). A proteolytic mechanism is now found to control ORE1 degradation, and thus senescence, during nitrogen starvation.
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.
P>The onset and progression of senescence are under genetic and environmental control. The Arabidopsis thaliana NAC transcription factor ANAC092 (also called AtNAC2 and ORE1) has recently been shown to control age-dependent senescence, but its mode of action has not been analysed yet. To explore the regulatory network administered by ANAC092 we performed microarray-based expression profiling using estradiol-inducible ANAC092 overexpression lines. Approximately 46% of the 170 genes up-regulated upon ANAC092 induction are known senescence-associated genes, suggesting that the NAC factor exerts its role in senescence through a regulatory network that includes many of the genes previously reported to be senescence regulated. We selected 39 candidate genes and confirmed their time-dependent response to enhanced ANAC092 expression by quantitative RT-PCR. We also found that the majority of them (24 genes) are up-regulated by salt stress, a major promoter of plant senescence, in a manner similar to that of ANAC092, which itself is salt responsive. Furthermore, 24 genes like ANAC092 turned out to be stage-dependently expressed during seed growth with low expression at early and elevated expression at late stages of seed development. Disruption of ANAC092 increased the rate of seed germination under saline conditions, whereas the opposite occurred in respective overexpression plants. We also detected a delay of salinity-induced chlorophyll loss in detached anac092-1 mutant leaves. Promoter-reporter (GUS) studies revealed transcriptional control of ANAC092 expression during leaf and flower ageing and in response to salt stress. We conclude that ANAC092 exerts its functions during senescence and seed germination through partly overlapping target gene sets.
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.
The Arabidopsis tandem-pore K+ (TPK) channels displaying four transmembrane domains and two pore regions share structural homologies with their animal counterparts of the KCNK family. In contrast to the Shaker-like Arabidopsis channels (six transmembrane domains/one pore region), the functional properties and the biological role of plant TPK channels have not been elucidated yet. Here, we show that AtTPK4 (KCO4) localizes to the plasma membrane and is predominantly expressed in pollen. AtTPK4 (KCO4) resembles the electrical properties of a voltage-independent K+ channel after expression in Xenopus oocytes and yeast. Hyperpolarizing as well as depolarizing membrane voltages elicited instantaneous K+ currents, which were blocked by extracellular calcium and cytoplasmic protons. Functional complementation assays using a K+ transport-deficient yeast confirmed the biophysical and pharmacological properties of the AtTPK4 channel. The features of AtTPK4 point toward a role in potassium homeostasis and membrane voltage control of the growing pollen tube. Thus, AtTPK4 represents a member of plant tandem-pore-K+ channels, resembling the characteristics of its animal counterparts as well as plant-specific features with respect to modulation of channel activity by acidosis and calcium
Oxidative stress causes dramatic changes in the expression levels of many genes. The formation of a functional protein through successful mRNA translation is central to a coordinated cellular response. To what extent the response towards reactive oxygen species (ROS) is regulated at the translational level is poorly understood. Here we analysed leaf- and tissue-specific translatomes using a set of transgenic Arabidopsis thaliana lines expressing a FLAG-tagged ribosomal protein to immunopurify polysome-bound mRNAs before and after oxidative stress. We determined transcript levels of 171 ROS-responsive genes upon paraquat treatment, which causes formation of superoxide radicals, at the whole-organ level. Furthermore, the translation of mRNAs was determined for five cell types: mesophyll, bundle sheath, phloem companion, epidermal and guard cells. Mesophyll and bundle sheath cells showed the strongest response to paraquat treatment. Interestingly, several ROS-responsive transcription factors displayed cell type-specific translation patterns, while others were translated in all cell types. In part, cell type-specific translation could be explained by the length of the 5-untranslated region (5-UTR) and the presence of upstream open reading frames (uORFs). Our analysis reveals insights into the translational regulation of ROS-responsive genes, which is important to understanding cell-specific responses and functions during oxidative stress.
The study illustrates the response of different Arabidopsis thaliana leaf cells and tissues to oxidative stress at the translational level, an aspect of reactive oxygen species (ROS) biology that has been little studied in the past. Our data reveal insights into how translational regulation of ROS-responsive genes is fine-tuned at the cellular level, a phenomenon contributing to the integrated physiological response of leaves to stresses involving changes in ROS levels.
Application of crab shell chitin or pentamer chitin oligosaccharide to Arabidopsis seedlings increased tolerance to salinity in wild-type but not in knockout mutants of the LysM Receptor-Like Kinase1 (CERK1/LysM RLK1) gene, known to play a critical role in signaling defense responses induced by exogenous chitin. Arabidopsis plants overexpressing the endochitinase chit36 and hexoaminidase excy1 genes from the fungus Trichoderma asperelleoides T203 showed increased tolerance to salinity, heavy-metal stresses, and Botrytis cinerea infection. Resistant lines, overexpressing fungal chitinases at different levels, were outcrossed to lysm rlk1 mutants. Independent homozygous hybrids lost resistance to biotic and abiotic stresses, despite enhanced chitinase activity. Expression analysis of 270 stress-related genes, including those induced by reactive oxygen species (ROS) and chitin, revealed constant up-regulation (at least twofold) of 10 genes in the chitinase-overexpressing line and an additional 76 salt-induced genes whose expression was not elevated in the lysm rlk1 knockout mutant or the hybrids harboring the mutation. These findings elucidate that chitin-induced signaling mediated by LysM RLK1 receptor is not limited to biotic stress response but also encompasses abiotic-stress signaling and can be conveyed by ectopic expression of chitinases in plants.
Since its discovery over two decades ago as an important cell death regulator in Arabidopsis thaliana, the role of LESION SIMULATING DISEASE 1 (LSD1) has been studied intensively within both biotic and abiotic stress responses as well as with respect to plant fitness regulation. However, its molecular mode of action remains enigmatic. Here, we demonstrate that nucleo-cytoplasmic LSD1 interacts with a broad range of other proteins that are engaged in various molecular pathways such as ubiquitination, methylation, cell cycle control, gametogenesis, embryo development and cell wall formation. The interaction of LSD1 with these partners is dependent on redox status, as oxidative stress significantly changes the quantity and types of LSD1-formed complexes. Furthermore, we show that LSD1 regulates the number and size of leaf mesophyll cells and affects plant vegetative growth. Importantly, we also reveal that in addition to its function as a scaffold protein, LSD1 acts as a transcriptional regulator. Taken together, our results demonstrate that LSD1 plays a dual role within the cell by acting as a condition-dependent scaffold protein and as a transcription regulator.
BackgroundPachycladon cheesemanii is a close relative of Arabidopsis thaliana and is an allotetraploid perennial herb which is widespread in the South Island of New Zealand. It grows at altitudes of up to 1000m where it is subject to relatively high levels of ultraviolet (UV)-B radiation. To gain first insights into how Pachycladon copes with UV-B stress, we sequenced its genome and compared the UV-B tolerance of two Pachycladon accessions with those of two A. thaliana accessions from different altitudes.ResultsA high-quality draft genome of P. cheesemanii was assembled with a high percentage of conserved single-copy plant orthologs. Synteny analysis with genomes from other species of the Brassicaceae family found a close phylogenetic relationship of P. cheesemanii with Boechera stricta from Brassicaceae lineage I. While UV-B radiation caused a greater growth reduction in the A. thaliana accessions than in the P. cheesemanii accessions, growth was not reduced in one P. cheesemanii accession. The homologues of A. thaliana UV-B radiation response genes were duplicated in P. cheesemanii, and an expression analysis of those genes indicated that the tolerance mechanism in P. cheesemanii appears to differ from that in A. thaliana.ConclusionAlthough the P. cheesemanii genome shows close similarity with that of A. thaliana, it appears to have evolved novel strategies allowing the plant to tolerate relatively high UV-B radiation.
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.
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.
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 mu L
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 mu L - 100 mu L). 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.
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.
SKOR and GORK are outward-rectifying plant potassium channels from Arabidopsis thaliana. They belong to the Shaker superfamily of voltage-dependent K+ channels. Channels of this class are composed of four alpha-subunits and subunit assembly is a prerequisite for channel function. In this study the assembly mechanism of SKOR was investigated using the yeast two-hybrid system and functional assays in Xenopus oocytes and in yeast. We demonstrate that SKOR and GORK physically interact and assemble into heteromeric K-out channels. Deletion mutants and chimeric proteins generated from SKOR and the K-in channel alpha-subunit KAT1 revealed that the cytoplasmic C-terminus of SKOR determines channel assembly. Two domains thatchannel a-subunit KAT1 revealed that the cytoplasmic C-terminus of SKOR determines channel assembly. Two domains that are crucial for channel assembly were identified: i), a proximal interacting region comprising a putative cyclic nucleotide-binding domain together with 33 amino acids just upstream of this domain, and ii), a distal interacting region showing some resemblance to the K-T domain of KAT1. Both regions contributed differently to channel assembly. Whereas the proximal interacting region was found to be active on its own, the distal interacting region required an intact proximal interacting region to be active. K-out alpha-subunits did not assemble with K-in alpha-subunits because of the absence of interaction between their assembly sites