Arabidopsis ILITHYIA protein is necessary for proper chloroplast biogenesis and root development independent of eIF2α phosphorylation.

One of the main mechanisms blocking translation after stress situations is mediated by phosphorylation of the α-subunit of the eukaryotic initiation factor 2 (eIF2), performed in Arabidopsis by the protein kinase GCN2 which interacts and is activated by ILITHYIA(ILA). ILA is involved in plant immunity and its mutant lines present phenotypes not shared by the gcn2 mutants. The functional link between these two genes remains elusive in plants. In this study, we show that, although both ILA and GCN2 genes are necessary to mediate eIF2α phosphorylation upon treatments with the aromatic amino acid biosynthesis inhibitor glyphosate, their mutants develop distinct root and chloroplast phenotypes. Electron microscopy experiments reveal that ila mutants, but not gcn2, are affected in chloroplast biogenesis, explaining the macroscopic phenotype previously observed for these mutants. ila3 mutants present a complex transcriptional reprogramming affecting defense responses, photosynthesis and protein folding, among others. Double mutant analyses suggest that ILA has a distinct function which is independent of GCN2 and eIF2α phosphorylation. These results suggest that these two genes may have common but also distinct functions in Arabidopsis.

Short review: Metabolism of theFusarium mycotoxins deoxynivalenol and zearalenone in plants.

Plants have a high capacity to transform and thereby detoxify deleterious or poisonous compounds, like mycotoxins. The formation of glucose conjugates has a central role in this process. Mammals, however, are able to (partly) release the precursor substances during digestion, reactivating the mycotoxins. This short review provides a brief summary about the metabolism of theFusarium mycotoxins deoxynivalenol and zearalenone in plants. Two examples are discussed in greater detail. First, the formation of deoxynivalenol-3-glucoside in wheat is linked to a quantitative trait locus that is often used forFusarium head blight resistance breeding. Secondly, the metabolism of zearalenone inArabidopsis thaliana results in at least 17 different metabolites, all of which are potentially hazardous for humans and animals.

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) determination of phase II metabolites of the mycotoxin zearalenone in the model plant Arabidopsis thaliana.

The biotransformation products of zearalenone, a Fusarium mycotoxin, were elucidated using the model plant Arabidopsis thaliana. After treatment of plant seedlings with 50 microM zearalenone, both the liquid media and the plant extracts were analysed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). An array of 17 different metabolites, most prominently glucosides, malonylglucosides, di-hexose- and hexose-pentose disaccharides of zearalenone, and alpha- and beta-zearalenol, were detected in the samples. Time courses for the different zearalenone metabolites were recorded and they give a closer insight into the metabolism kinetics. A scheme proposing the zearalenone metabolism in A. thaliana is given. The aspect of food safety regarding the (potential) occurrence of masked mycotoxins in agricultural commodities is discussed.

Trichome distribution in Arabidopsis thaliana and its close relative Arabidopsis lyrata: molecular analysis of the candidate gene GLABROUS1.

GLABROUS1 (GL1) belongs to the large family of MYB transcription factors and is known to play a central role in trichome initiation. We studied trichome distribution and the molecular variation of GL1 in 28 A. thaliana accessions. Trichome density on rosette leaves was highly variable among those accessions. On the molecular level, we detected substantial sequence variation in a 3-kb fragment which included the complete coding region of the GL1 locus (pi = 0.01). Phylogenetic analysis of GL1 indicates the presence of two diverged clades among 28 accessions. Using ANOVA, we show that the phenotypic variation in trichome density cannot be explained by the sequence divergence between the two phylogenetic lineages. Sequence analysis of wild-type Arabidopsis thaliana and Arabidopsis lyrata accessions indicates that all amino acid substitutions are located outside of the conserved helix-turn-helix DNA-binding domains R2 and R3. Using plants of A. thaliana and A. lyrata with either naturally occurring or ethyl methane sulfonate--induced glabrous phenotypes, we demonstrate that the last 14 C-terminal amino acids of the GL1 gene have no major impact on the initiation of trichomes.

Cloning and expression of cDNAs encoding alpha1,3-fucosyltransferase homologues from Arabidopsis thaliana.

The core alpha1,3-fucosyltransferases are involved in the synthesis of glycans specific to plants and invertebrates which are known to be immunogenic and allergenic. We report the identification, isolation and characterisation of the cDNAs of three genes (FucTA, FucTB and FucTC) encoding proteins similar to alpha1,3-fucosyltransferases in Arabidopsis thaliana. Reverse transcription-polymerase chain reaction was used to amplify the full length coding sequence of FucTA. The FucTA gene, which consists of seven exons, encodes a presumptive protein of 501 amino acids showing an overall sequence identity of 66% to the protein encoded by the recently isolated mung bean Fuc-T C3 cDNA. FucTA was expressed in Pichia pastoris under the control of the AOX1 gene promoter. The soluble enzyme was found to catalyse the same reaction as mung bean core alpha1,3-fucosyltransferase as judged by analyses of the products by MALDI-TOF and high-performance liquid chromatography. The FucTB cDNA was isolated from a lambda-ZAP library, but the clone used an alternative splicing site between the second and third exon resulting in a premature stop codon. The FucTC gene encodes a protein with less than 40% identity to FucTA across 115 amino acids of a total of 401 amino acids and is a member of a new sub-family of plant alpha1,3/4-fucosyltransferase homologues.

The SHORT-ROOT gene controls radial patterning of the Arabidopsis root through radial signaling.

Asymmetric cell divisions play an important role in the establishment and propagation of the cellular pattern of plant tissues. The SHORT-ROOT (SHR) gene is required for the asymmetric cell division responsible for formation of ground tissue (endodermis and cortex) as well as specification of endodermis in the Arabidopsis root. We show that SHR encodes a putative transcription factor with homology to SCARECROW (SCR). From analyses of gene expression and cell identity in genetically stable and unstable alleles of shr, we conclude that SHR functions upstream of SCR and participates in a radial signaling pathway. Consistent with a regulatory role in radial patterning, ectopic expression of SHR results in supernumerary cell divisions and abnormal cell specification in the root meristem.

Post-transcriptional control of the Arabidopsis auxin efflux carrier EIR1 requires AXR1.

The auxin efflux carrier EIR1 (also known as AGR and AtPIN2) is a key mediator of the response of Arabidopsis roots to gravity [1,2]. This response is thought to require the establishment of a transient auxin gradient in the root meristem, resulting in differential cell elongation [3]. Recent reports suggest that EIR1 is essential for the asymmetric distribution of auxin in the root meristem [4-7], but the regulatory aspects of this process are still not fully understood. Here, we studied the regulation of EIR1 in Arabidopsis using two reporters: one was a translational fusion that contained the entire EIR1 coding sequence, and the other a transcriptional fusion that had no EIR1 coding sequence. We found that EIR1 is controlled at the post-transcriptional level. The translational fusion was unstable in response to changes in auxin homeostasis, and was destabilized by cycloheximide. In contrast, the protein was stabilized in the axr1-3 mutant, which is auxin resistant and defective in auxin responses such as root gravitropism [8,9]. AXR1 is thought to participate in ubiquitin-mediated control of protein stability [10-12]. The dependence of EIR1 reporter expression on auxin concentrations and AXR1 suggests that auxin transport is regulated through a feedback regulatory loop that affects protein stability in response to auxin.

Nonradioactive labeling of large DNA fragments for genome walking, RFLP and northern blot analysis.

In this report, we present a simple nonradioactive labeling procedure for DNA fragments of high specific labeling density that can be used for a variety of applications. The protocol is based on the universal mono-functional platinum reagent for chemical digoxigenin (DIG) labeling of nucleic acids. The labeling protocol was optimized for large DNA templates as complete bacterial artificial chromosomes (BAC). Variations of incubation time and temperature improved the labeling density such that about 30% of the nucleotides were DIG-modified within 30 min. Furthermore, the refined procedure generates in a single-tube reaction and without prior digestion-labeled DNA fragments of 0.5-4.0 kb from a 130-kb template. Hybridization experiments were performed on Southern and northern blots and allowed the detection of single copy genes in 2.5 micrograms genomic DNA from Arabidopsis thaliana, which has a haploid genome size of 0.13 pg (ca. 120 Mb) and medium expressed transcripts from 0.8 microgram poly(A)+ RNA, respectively. The extremely high specific labeling density, the stability and the universal application of the probe generated with the platinum reagent makes this method a useful alternative to classical radioactive nuclei acids labeling techniques.

Generation of co-dominant PCR-based markers by duplex analysis on high resolution gels.

Rapid and efficient procedures for the detection of sequence polymorphisms are essential for chromosomal walking and mutation detection analyses. While DNA chip technology and denaturing high-performance liquid chromatography (DHPLC) are the methods of choice for large scale facilities, small laboratories are dependent on simple ready-to-use techniques. We show that heteroduplex analysis on high resolution gel matrices efficiently detects sequence polymorphism differing as little as a single base pair (e.g. single-nucleotide polymorphism, SNP) with standard laboratory equipment. Furthermore, the matrices also discerned differences between homoduplexes, a prerequisite for co-dominant markers. The markers thus generated are referred to as duplex analysis markers. We designed PCR primers for 36 Arabidopsis thaliana loci ranging in length from 230 bp to 1000 bp. Among three ecotypes, more than half (n = 19) of the loci examined were polymorphic; five of which contained three different alleles. This simple, high resolution technique can be used to rapidly convert sequence tagged sites into co-dominant PCR-based molecular markers for fine-scale mapping studies and chromosomal walking strategies as well as for the detection of mutations in particular genes.

Conditional root expansion mutants of Arabidopsis.

Regulation of cell expansion is essential to the formation of plant organs. We have characterized 21 mutations, representing six loci, that cause abnormal cell expansion in the root of Arabidopsis thaliana. The phenotype of these mutants is conditional upon the rate of root growth. Calculation of cell volumes indicated that the mutations resulted in defects in either the orientation or the extent of expansion or in both. Analysis of cortical microtubules in the mutants suggested that a shift in the orientation of cell expansion may not be dependent on a change in the orientation of the microtubules. Double mutant combinations resulted in loss of the conditional phenotype suggesting that the genes may act in a similar pathway or encode partially redundant functions.

The SABRE gene is required for normal cell expansion in Arabidopsis.

Regulated cell expansion is an important determinant of organ shape in higher plants. The sabre mutation results in abnormal cell expansion in Arabidopsis. There is a shift in the orientation of expansion evident primarily in root cortex cells. The SABRE gene has been cloned and found to encode a novel protein. Reduction of effective levels of the plant phytohormone ethylene through use of inhibitors and an insensitive mutant resulted in partial rescue of the sabre phenotype. This suggested that one of the roles of SABRE is to counter the action of ethylene in promoting radial expansion in plant cells.

Characterization of the signal recognition particle (SRP) RNA population of tomato (Lycopersicon esculentum).

Molecular cloning of 30 cDNAs and subsequent characterization of the corresponding SRP RNA from four cultivars of tomato (Lycopersicon esculentum) revealed altogether 14 sequence variants, which could be ordered into six groups. The expression of five representatives from these groups was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) in different cultivars and different tissues. Although one cultivar-specific SRP RNA variant could be detected in the leaf SRP RNA population, identical SRP RNA populations seem to be present in the four different cultivars as well as in different tissues, such as leaves, flowers, fruits, stems and roots. Sequence comparison revealed that several variants might have evolved by recombination of two different SRP RNA sequences. On the basis of five SRP RNA variants, the current secondary structure model was refined and a new conserved structural element was detected. Comparative sequence analysis of domain II from all known SRP RNA homologues reveals a remarkable conservation of this element. As demonstrated previously, the corresponding area overlaps with a region that interact with the SRPp68/p72 heterodimer and/or with ribosomes. Based on structural and functional considerations, we propose that the domain IV structure together with the highly conserved area of domain II constitutes the essential core of the SRP RNA.

Comparative evolutionary analysis of rDNA ITS regions in Drosophila.

The internal transcribed spacer (ITS) of the ribosomal DNA is generally considered to be under low functional constraint, and it is therefore often treated as a typical nonfunctional spacer sequence. We have analyzed the ITS regions of five species from the Drosophila melanogaster subgroup, two Drosophila species from outside this group (D. pseudoobscura and D. virilis), as well as from the more distantly related dipteran fly Musca domestica. The sequence comparisons show a distinctive conservation/divergence pattern, indicating that some regions are more conserved than others. Moreover, secondary-structure calculations indicate several conserved structural elements within the ITS regions. On the other hand, a statistical test that allows us to estimate the fraction of sites that are not under selective constraint suggests that more than half of the spacer is apparently free to diverge and evolves with a rate that is close to the neutral rate of sequence evolution in Drosophila. The ITS sequences can be used to derive a molecular phylogeny for the species under study. We find that the ITS tree is largely in line with the so-far-known phylogeny of this group of species, with one difference. The species most distant within the D. melanogaster subgroup is D. yakuba, rather than D. orena, as is normally assumed.

Root development in Arabidopsis: four mutants with dramatically altered root morphogenesis.

A genetic analysis of root development in Arabidopsis thaliana has identified mutants that have abnormal morphogenesis. Four of these root morphogenesis mutants show dramatic alterations in post-embryonic root development. The short-root mutation results in a change from indeterminate to determinate root growth and the loss of internal root cell layers. The cobra and lion's tail mutations cause abnormal root cell expansion which is conditional upon the rate of root growth. Expansion is greatest in the epidermal cells in cobra and in the stele cells in lion's tail. The sabre mutation causes abnormal cell expansion that is greatest in the root cortex cell layer and is independent of the root growth rate. The tissue-specific effects of these mutations were characterized with monoclonal antibodies and a transgenic marker line. Genetic combinations of the four mutants have provided insight into the regulation of growth and cell shape during Arabidopsis root development.