We used four mutants having albino or pale green phenotypes with disrupted nuclear-encoded chloroplast proteins to analyze the regulatory system of metabolites in chloroplast. We performed an integrated analyses of transcriptomes and metabolomes of the four mutants. Transcriptome analysis was carried out using the Agilent Arabidopsis 2 Oligo Microarray, and metabolome analysis with two mass spectrometers; a direct-infusion Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR/MS) and a gas chromatograph-time of flight mass spectrometer. Among approximately 200 known metabolites detected by the FT-ICR/MS, 71 metabolites showed significant changes in the mutants when compared with controls (Ds donor plants). Significant accumulation of several amino acids (glutamine, glutamate and asparagine) was observed in the albino and pale green mutants. Transcriptome analysis revealed altered expressions of genes in several metabolic pathways. For example, genes involved in the tricarboxylic acid cycle, the oxidative pentose phosphate pathway, and the de novo purine nucleotide biosynthetic pathway were up-regulated. These results suggest that nitrogen assimilation is constitutively promoted in the albino and pale green mutants. The accumulation of ammonium ions in the albino and pale green mutants was consistently higher than in Ds donor lines. Furthermore, genes related to pyridoxin accumulation and the de novo purine nucleotide biosynthetic pathway were up-regulated, which may have occurred as a result of the accumulation of glutamine in the albino and pale green mutants. The difference in metabolic profiles seems to be correlated with the disruption of chloroplast internal membrane structures in the mutants. In albino mutants, the alteration of metabolites accumulation and genes expression is stronger than pale green mutants.
Electronic supplementary material
The online version of this article (doi:10.1007/s11103-014-0194-9) contains supplementary material, which is available to authorized users.
Albino or pale-green; Arabidopsis thaliana; Chloroplast; Metabolome; Transcriptome; Nitrogen assimilation
An analysis of the salinity tolerance of 354 Arabidopsis thaliana accessions showed that some accessions were more tolerant to salt shock than the reference accession, Col-0, when transferred from 0 to 225 mM NaCl. In addition, several accessions, including Zu-0, showed marked acquired salt tolerance after exposure to moderate salt stress. It is likely therefore that Arabidopsis plants have at least two types of tolerance, salt shock tolerance and acquired salt tolerance. To evaluate a role of well-known salt shock tolerant gene SOS1 in acquired salt tolerance, we isolated a sos1 mutant from ion-beam-mutagenized Zu-0 seedlings. The mutant showed severe growth inhibition under salt shock stress owing to a single base deletion in the SOS1 gene and was even more salt sensitive than Col-0. Nevertheless, it was able to survive after acclimation on 100 mM NaCl for 7 d followed by 750 mM sorbitol for 20 d, whereas Col-0 became chlorotic under the same conditions. We propose that genes for salt acclimation ability are different from genes for salt shock tolerance and play an important role in the acquisition of salt or osmotic tolerance.
salt tolerance; Arabidopsis; natural variation; salt acclimation
AtALMT1 (Arabidopsis thaliana ALuminum activated Malate Transporter 1) encodes an Arabidopsis thaliana malate transporter that has a pleiotropic role in Arabidopsis stress tolerance. Malate released through AtALMT1 protects the root tip from Al rhizotoxicity, and recruits beneficial rhizobacteria that induce plant immunity. To examine whether the overexpression of AtALMT1 can improve these traits, the gene, driven by the cauliflower mosaic virus 35S promoter, was introduced into the Arabidopsis ecotype Columbia. Overexpression of the gene enhanced both Al-activated malate excretion and the recruitment of beneficial bacteria Bacillus subtilis strain FB17. These findings suggest that overexpression of AtALMT1 can be used as an approach to enhance a plant's ability to release malate into the rhizosphere, which can enhance plant tolerance to some environmental stress factors.
Arabidopsis thaliana; AtALMT1; Bacillus subtilisstrain FB17; Pseudomonas syringae pv tomato DC3000; aluminum; malate exudation
Plants respond to dehydration stress and tolerate water-deficit status through complex physiological and cellular processes. Many genes are induced by water deficit. Abscisic acid (ABA) plays important roles in tolerance to dehydration stress by inducing many stress genes. ABA is synthesized de novo in response to dehydration. Most of the genes involved in ABA biosynthesis have been identified, and they are expressed mainly in leaf vascular tissues. Of the products of such genes, 9-cis-epoxycarotenoid dioxygenase (NCED) is a key enzyme in ABA biosynthesis. One of the five NCED genes in Arabidopsis, AtNCED3, is significantly induced by dehydration. To understand the regulatory mechanism of the early stages of the dehydration stress response, it is important to analyse the transcriptional regulatory systems of AtNCED3. In the present study, we found that an overlapping G-box recognition sequence (5′-CACGTG-3′) at −2248 bp from the transcriptional start site of AtNCED3 is an important cis-acting element in the induction of the dehydration response. We discuss the possible transcriptional regulatory system of dehydration-responsive AtNCED3 expression, and how this may control the level of ABA under water-deficit conditions.
Arabidopsis; abscisic acid (ABA); 9-cis-epoxycarotenoid dioxygenase (NCED); AtNCED3; dehydration response; cis-acting element
Arabidopsis belongs to the Brassicaceae family and plays an important role as a model plant for which researchers have developed fine-tuned genome resources. Genome sequencing projects have been initiated for other members of the Brassicaceae family. Among these projects, research on Chinese cabbage (Brassica rapa subsp. pekinensis) started early because of strong interest in this species. Here, we report the development of a library of Chinese cabbage full-length cDNA clones, the RIKEN BRC B. rapa full-length cDNA (BBRAF) resource, to accelerate research on Brassica species. We sequenced 10 000 BBRAF clones and confirmed 5476 independent clones. Most of these cDNAs showed high homology to Arabidopsis genes, but we also obtained more than 200 cDNA clones that lacked any sequence homology to Arabidopsis genes. We also successfully identified several possible candidate marker genes for plant defence responses from our analysis of the expression of the Brassica counterparts of Arabidopsis marker genes in response to salicylic acid and jasmonic acid. We compared gene expression of these markers in several Chinese cabbage cultivars. Our BBRAF cDNA resource will be publicly available from the RIKEN Bioresource Center and will help researchers to transfer Arabidopsis-related knowledge to Brassica crops.
Arabidopsis; Brassica rapa; full-length cDNA; jasmonic acid; salicylic acid
Thellungiella halophila (also known as T. salsuginea) is a model halophyte with a small size, short life cycle, and small genome. Thellungiella genes exhibit a high degree of sequence identity with Arabidopsis genes (90% at the cDNA level). We previously generated a full-length enriched cDNA library of T. halophila from various tissues and from whole plants treated with salinity, chilling, freezing stress, or ABA. We determined the DNA sequences of 20 000 cDNAs at both the 5'- and 3' ends, and identified 9569 distinct genes.
Here, we completely sequenced 1047 Thellungiella full-length cDNAs representing abiotic-stress-related genes, transcription factor genes, and protein phosphatase 2C genes. The predicted coding sequences, 5'-UTRs, and 3'-UTRs were compared with those of orthologous genes from Arabidopsis for length, sequence similarity, and structure. The 5'-UTR sequences of Thellungiella and Arabidopsis orthologs shared a significant level of similarity, although the motifs were rearranged. While examining the stress-related Thellungiella coding sequences, we found a short splicing variant of T. halophila salt overly sensitive 1 (ThSOS1), designated ThSOS1S. ThSOS1S contains the transmembrane domain of ThSOS1 but lacks the C-terminal hydrophilic region. The expression level of ThSOS1S under normal growth conditions was higher than that of ThSOS1. We also compared the expression levels of Na+-transport-system genes between Thellungiella and Arabidopsis by using full-length cDNAs from each species as probes. Several genes that play essential roles in Na+ excretion, compartmentation, and diffusion (SOS1, SOS2, NHX1, and HKT1) were expressed at higher levels in Thellungiella than in Arabidopsis.
The full-length cDNA sequences obtained in this study will be essential for the ongoing annotation of the Thellungiella genome, especially for further improvement of gene prediction. Moreover, they will enable us to find splicing variants such as ThSOS1S (AB562331).
Many accessions (ecotypes) of Arabidopsis have been collected. Although few differences exist among their nucleotide sequences, these subtle differences induce large genetic variation in phenotypic traits such as stress tolerance and flowering time. To understand the natural variability in salt tolerance, large-scale soil pot experiments were performed to evaluate salt tolerance among 350 Arabidopsis thaliana accessions. The evaluation revealed a wide variation in the salt tolerance among accessions. Several accessions, including Bu-5, Bur-0, Ll-1, Wl-0, and Zu-0, exhibited marked stress tolerance compared with a salt-sensitive experimental accession, Col-0. The salt-tolerant accessions were also evaluated by agar plate assays. The data obtained by the large-scale assay correlated well with the results of a salt acclimation (SA) assay, in which plants were transferred to high-salinity medium following placement on moderate-salinity medium for 7 d. Genetic analyses indicated that the salt tolerance without SA is a quantitative trait under polygenic control, whereas salt tolerance with SA is regulated by a single gene located on chromosome 5 that is common among the markedly salt-tolerant accessions. These results provide important information for understanding the mechanisms underlying natural variation of salt tolerance in Arabidopsis.
Acclimation; Arabidopsis thaliana accessions; mapping; natural variation; salt stress
The National BioResource Project (NBRP) is a Japanese project that aims to establish a system for collecting, preserving and providing bioresources for use as experimental materials for life science research. It is promoted by 27 core resource facilities, each concerned with a particular group of organisms, and by one information center. The NBRP database is a product of this project. Thirty databases and an integrated database-retrieval system (BioResource World: BRW) have been created and made available through the NBRP home page (http://www.nbrp.jp). The 30 independent databases have individual features which directly reflect the data maintained by each resource facility. The BRW is designed for users who need to search across several resources without moving from one database to another. BRW provides access to a collection of 4.5-million records on bioresources including wild species, inbred lines, mutants, genetically engineered lines, DNA clones and so on. BRW supports summary browsing, keyword searching, and searching by DNA sequences or gene ontology. The results of searches provide links to online requests for distribution of research materials. A circulation system allows users to submit details of papers published on research conducted using NBRP resources.
Inhibitors of apoptosis proteins (IAPs) interact with caspases and inhibit their protease activity, whereas the IAP-inhibitory proteins Smac/DIABLO in mammals and Reaper, Hid, and Grim in flies relieve IAP-mediated inhibition [1-5] to induce cell death. Here we describe the functional characterization of the novel Drosophila cell death protein Sickle (Skl), which binds to IAPs and neutralizes their apoptotic inhibitory activity. Skl exhibits no sequence homology to Reaper, Hid, Grim, or Smac/DIABLO, except within the 4 residue N-terminal IAP binding motif. Skl interacts with Drosophila and mammalian IAPs and can promote caspase activation in the presence of IAPs. Consistent with these findings, expression of Skl in Drosophila and mammalian cell lines or in Drosophila embryos induces apoptosis. Skl can also synergize with Grim to induce cell death in the Drosophila eye imaginal disc. Based on biochemical and structural data, the N terminus of Skl, like that of the mammalian Smac/ DIABLO, is absolutely required for its apoptotic and caspase-promoting activities and its ability to interact with IAPs. These findings point to conservation in the structure and function of the IAP-inhibitory proteins across species and suggest the existence of other family members.
The western flower thrips (Frankliniella occidentalis [Pergande]) is one of the most important insect herbivores of cultivated plants. However, no pesticide provides complete control of this species, and insecticide resistance has emerged around the world. We previously reported the important role of jasmonate (JA) in the plant's immediate response to thrips feeding by using an Arabidopsis leaf disc system. In this study, as the first step toward practical use of JA in thrips control, we analyzed the effect of JA-regulated Arabidopsis defense at the whole plant level on thrips behavior and life cycle at the population level over an extended period. We also studied the effectiveness of JA-regulated plant defense on thrips damage in Chinese cabbage (Brassica rapa subsp. pekinensis).
Thrips oviposited more on Arabidopsis JA-insensitive coi1-1 mutants than on WT plants, and the population density of the following thrips generation increased on coi1-1 mutants. Moreover, thrips preferred coi1-1 mutants more than WT plants. Application of JA to WT plants before thrips attack decreased the thrips population. To analyze these important functions of JA in a brassica crop plant, we analyzed the expression of marker genes for JA response in B. rapa. Thrips feeding induced expression of these marker genes and significantly increased the JA content in B. rapa. Application of JA to B. rapa enhanced plant resistance to thrips, restricted oviposition, and reduced the population density of the following generation.
Our results indicate that the JA-regulated plant defense restricts thrips performance and preference, and plays an important role in the resistance of Arabidopsis and B. rapa to thrips damage.
Development of a multicellular organism requires precise coordination of cell division and cell type determination. The selector homeoprotein Even skipped (Eve) plays a very specific role in determining cell identity in the Drosophila embryo, both during segmentation and in neuronal development. However, studies of gene expression in eve mutant embryos suggest that eve regulates the embryonic expression of the vast majority of genes. We present here genetic interaction and phenotypic analysis showing that eve functions in the trol pathway to regulate the onset of neuroblast division in the larval CNS. Surprisingly, Eve is not detected in the regulated neuroblasts, and culture experiments reveal that Eve is required in the body, not the CNS. Furthermore, the effect of an eve mutation can be rescued both in vivo and in culture by the hormone ecdysone. These results suggest that eve is required to produce a trans-acting factor that stimulates cell division in the larval brain.
eve; trol; Ecdysone; Neuroblast; Proliferation; Drosophila
Groucho acts as a co-repressor for several Drosophila DNA binding transcriptional repressors. Several of these proteins have been found to contain both Groucho-dependent and - independent repression domains, but the extent to which this distinction has functional consequences for the regulation of different target genes is not known. The product of the pair-rule gene even skipped has previously been shown to contain a Groucho-independent repression activity. In the Even skipped protein, outside the Groucho-independent repression domain, we have identified a conserved C-terminal motif (LFKPY), similar to motifs that mediate Groucho interaction in Hairy, Runt and Hückebein. Even skipped interacts with Groucho in yeast and in vitro, and groucho and even skipped genetically interact in vivo. Even skipped with a mutated Groucho interaction motif, which abolished binding to Groucho, showed a significantly reduced ability to rescue the even skipped null phenotype when driven by the complete even skipped regulatory region. Replacing this motif with a heterologous Groucho interaction motif restored the rescuing function of Even skipped in segmentation. Further functional assays demonstrated that the Even skipped C terminus acts as a Groucho-dependent repression domain in early Drosophila embryos. This novel repression domain was active on two target genes that are normally repressed by Even skipped at different concentrations, paired and sloppy paired. When the Groucho interaction motif is mutated, repression of each target gene is reduced to a similar extent, with some activity remaining. Thus, the ability of Even skipped to repress different target genes at different concentrations does not appear to involve differential recruitment or function of Groucho. The accumulation of multiple domains of similar function within a single protein may be a common evolutionary mechanism that fine-tunes the level of activity for different regulatory functions.
Repressor; Co-repressor; Embryogenesis; Segmentation; Drosophila
Engrailed is a key transcriptional regulator in the nervous system and in the maintenance of developmental boundaries in Drosophila, and its vertebrate homologs regulate brain and limb development. Here, we show that the functions of both of the Hox cofactors Extradenticle and Homothorax play essential roles in repression by Engrailed. Mutations that remove either of them abrogate the ability of Engrailed to repress its target genes in embryos, both cofactors interact directly with Engrailed, and both stimulate repression by Engrailed in cultured cells. We suggest a model in which Engrailed, Extradenticle and Homothorax function as a complex to repress Engrailed target genes. These studies expand the functional requirements for extradenticle and homothorax beyond the Hox proteins to a larger family of non-Hox homeodomain proteins.
Drosophila; Embryogenesis; Homeodomain; Transcription; Repressor; Cofactor; Pbx; Meis
BRCA2 is a breast tumour susceptibility factor with functions in maintaining genome stability through ensuring efficient double-strand DNA break (DSB) repair via homologous recombination. Although best known in vertebrates, fungi, and higher plants also possess BRCA2-like genes. To investigate the role of Arabidopsis BRCA2 genes in DNA repair in somatic cells, transposon insertion mutants of the AtBRCA2a and AtBRCA2b genes were identified and characterized. atbrca2a-1 and atbrca2b-1 mutant plants showed hypersensitivity to genotoxic stresses compared to wild-type plants. An atbrca2a-1/atbrca2b-1 double mutant showed an additive increase in sensitivity to genotoxic stresses compared to each single mutant. In addition, it was found that atbrca2 mutant plants displayed fasciation and abnormal phyllotaxy phenotypes with low incidence, and that the ratio of plants exhibiting these phenotypes is increased by γ-irradiation. Interestingly, these phenotypes were also induced by γ-irradiation in wild-type plants. Moreover, it was found that shoot apical meristems of the atbrca2a-1/atbrca2b-1 double mutant show altered cell cycle progression. These data suggest that inefficient DSB repair in the atbrca2a-1/atbrca2b-1 mutant leads to disorganization of the programmed cell cycle of apical meristems.
Arabidopsis; BRCA2; cell cycle; double-strand DNA breaks; fasciation; homologous recombination; γ-ray
Alternative splicing (AS) is a mechanism by which multiple types of mature mRNAs are generated from a single pre-mature mRNA. In this study, we completely sequenced 1800 full-length cDNAs from Arabidopsis thaliana, which had 5′ and/or 3′ sequences that were previously found to have AS events or alternative transcription start sites. Unexpectedly, these sequences gave us further evidence of AS, as 601 out of 1800 transcripts showed novel AS events. We focused on the combination patterns of multiple AS events within individual genes. Interestingly, some specific AS event combination patterns tended to appear more frequently than expected. The two most common patterns were: (i) alternative donor–0∼12 times of exon skips–alternative acceptor and (ii) several times (∼8) of retained introns. We also found that multiple AS events in a transcript tend to have the same effects concerning the length of the mature mRNA. Our current results are consistent with our previous observations, which showed changes in AS profiles under different conditions, and suggest the involvement of hypothetical cis- and trans-acting factors in the regulation of AS events.
Arabidopsis; alternative splicing; bioinformatics; full-length cDNA
Insect feeding retards plant growth and decreases crop productivity. Plants respond to insect feeding at the molecular, cellular and physiological levels. The roles of the plant hormones jasmonic acid (JA), ethylene (ET) and salicylic acid (SA) in plant responses to insect feeding have been studied. However, these studies are focused on the plant responses to feeding by well-studied caterpillar type insects or aphid pests. In contrast, we have focused on a minute insect pest, the western flower thrips (Frankliniella occidentalis). Analyses of the responses of hormone-related mutants of Arabidopsis (i.e., JA-insensitive mutant coi1-1, ET-insensitive mutants ein2-1 and ein3-1, and SA-deficient mutant eds16-1) and transcriptome-based comparative analyses indicate the central role of JA in plant responses to thrips feeding. Our work clearly shows that JA signaling, but not JA/ET signaling, is involved in plant tolerance to thrips feeding. We intend to examine the utility and suitability of the Arabidopsis-thrips system in studies of plant responses to insect feeding.
Arabidopsis thaliana; ethylene; Frankliniella occidentalis; insect feeding; jasmonate; western flower thrips
Under acid soil condition, rhizotoxic ions such as aluminum and protons cause severe yield loss in food and biomass production via inhibition of root growth and/or enhancement of sensitivity to drought stress. Therefore, improvement of crop tolerance to rhizotoxic ions would be an important target for crop breeding, and identification of key genes that regulate tolerance to both aluminum and proton ions should provide a solution to this limitation. We recently isolated a mutant that shows hyper-sensitivity to protons in root growth, namely stop1 (sensitive to proton rhizotoxicity). The stop1 mutant was isolated from an ethyl methanesulfonate mutagenized population by measuring root growth under low pH (pH 4.3). Interestingly, stop1 also shows hypersensitivity to aluminum ion, but not to other rhizotoxic ions. Cloning of the STOP1 gene revealed that it encodes a Cys2/His2 zinc-finger type transcription factor, and a conserved His residue was replaced with Tyr in the predicted amino acid sequence for the mutant gene. This indicates that STOP1 is involved in the signal transduction pathway of proton and aluminum tolerance. Indeed, under acidic condition, stop1 failed to induce the AtALMT1 gene, which encodes one of the major factors for aluminum tolerance. By searching a public database, we have identified genes homologous to STOP1 in rice, maize and some other plants. Thus, the STOP1 family genes may act as a key factor for acid tolerance in a wide variety of plants.
proton-rhizotoxicity; Arabidopsis thaliana; STOP (Sensitive TO Proton rhizotoxicity); Cys2/His2 type zinc-finger protein; aluminum toxicity,
DNA supercoiling factor (SCF) was first identified in silkworm as a protein that generates negative supercoils in DNA in conjunction with eukaryotic topoisomerase II. To analyze the in vivo role of the factor, we cloned a cDNA encoding Drosophila melanogaster SCF. Northern analysis revealed 1.6- and 1.8-kb mRNAs throughout development. The longer mRNA contains an open reading frame that shares homology with mouse reticulocalbin whereas the shorter one encodes a truncated version lacking the N-terminal signal peptide-like sequence. An antibody against SCF detected a 45-kDa protein in the cytoplasmic fraction and a 30-kDa protein in the nuclear fraction of embryonic extracts. Immunoprecipitation suggests that the 30-kDa protein interacts with topoisomerase II in the nucleus, and hence that it is a functional form of SCF. Immunostaining of blastoderm embryos showed that SCF is present in nuclei during interphase but is excluded from mitotic chromosomes. In larvae, the antibody stained the nuclei of several tissues including a posterior part of the salivary gland. This latter staining was associated with natural or ecdysteroid-induced puffs on polytene chromosomes. Upon heat treatment of larvae, the staining on the endogenous puffs disappeared, and strong staining appeared on heat shock puffs. These results implicate SCF in gene expression.
Active transcriptional repression has been characterized as a function of many regulatory factors. It facilitates combinatorial regulation of gene expression by allowing repressors to be dominant over activators under certain conditions. Here, we show that the Engrailed protein uses two distinct mechanisms to repress transcription. One activity is predominant under normal transient transfection assay conditions in cultured cells. A second activity is predominant in an in vivo active repression assay. The domain mediating the in vivo activity (eh1) is highly conserved throughout several classes of homeoproteins and interacts specifically with the Groucho corepressor. While eh1 shows only weak activity in transient transfections, much stronger activity is seen in culture when an integrated target gene is used. In this assay, the relative activities of different repression domains closely parallel those seen in vivo, with eh1 showing the predominant activity. Reducing the amounts of repressor and target gene in a transient transfection assay also increases the sensitivity of the assay to the Groucho interaction domain, albeit to a lesser extent. This suggests that it utilizes rate-limiting components that are relatively low in abundance. Since Groucho itself is abundant in these cells, the results suggest that a limiting component is recruited effectively by the repressor-corepressor complex only on integrated target genes.