The TASTY locus on chromosome 1 of Arabidopsis affects feeding of the insect herbivore Trichoplusia ni

The generalist insect herbivore Trichoplusia ni (cabbage looper) readily consumes Arabidopsis and can complete its entire life cycle on this plant. Natural isolates (ecotypes) of Arabidopsis are not equally susceptible to T. ni feeding. While some are hardly touched by T. ni, others are eaten completely to the ground. Comparison of two commonly studied Arabidopsis ecotypes in choice experiments showed that Columbia is considerably more resistant than Landsberg erecta. In no-choice experiments, where larvae were confined on one or the other ecotype, weight gain was more rapid on Landsberg erecta than on Columbia. Genetic mapping of this difference in insect susceptibility using recombinant inbred lines resulted in the discovery of the TASTY locus near 85 cM on chromosome 1 of Arabidopsis. The resistant allele of this locus is in the Columbia ecotype, and an F(1) hybrid has a sensitive phenotype that is similar to that of Landsberg erecta. The TASTY locus is distinct from known genetic differences between Columbia and Landsberg erecta that affect glucosinolate content, trichome density, disease resistance, and flowering time.     

Analysis of naturally occurring late flowering in Arabidopsis thaliana

Summary We have examined the late-flowering behavior of two ecotypes of Arabidopsis thaliana, Sf-2 and Le-0. The late-flowering trait segregates as a single dominant gene in crosses with the early-flowering Columbia ecotype. This gene, which we refer to as FLA, is located at one end of chromosome 4 between RFLP markers 506 and 3843 and is thus distinct from previously mapped genes that affect flowering time. The extreme delay in flowering time caused by the FLA gene can be overcome by vernalization in both the ecotypes in which it occurs naturally and in the Columbia ecotype into which this gene has been introgressed.     

Genetic regulation of gene expression during shoot development in Arabidopsis

The genetic control of gene expression during shoot development in Arabidopsis thaliana was analyzed by combining quantitative trait loci (QTL) and microarray analysis. Using oligonucleotide array data from 30 recombinant inbred lines derived from a cross of Columbia and Landsberg erecta ecotypes, the Arabidopsis genome was scanned for marker-by-gene linkages or so-called expression QTL (eQTL). Single-feature polymorphisms (SFPs) associated with sequence disparities between ecotypes were purged from the data. SFPs may alter the hybridization efficiency between cDNAs from one ecotype with probes of another ecotype. In genome scans, five eQTL hot spots were found with significant marker-by-gene linkages. Two of the hot spots coincided with classical QTL conditioning shoot regeneration, suggesting that some of the heritable gene expression changes observed in this study are related to differences in shoot regeneration efficiency between ecotypes. Some of the most significant eQTL, particularly those at the shoot regeneration QTL sites, tended to show cis-chromosomal linkages in that the target genes were located at or near markers to which their expression was linked. However, many linkages of lesser significance showed expected "trans-effects," whereby a marker affects the expression of a target gene located elsewhere on the genome. Some of these eQTL were significantly linked to numerous genes throughout the genome, suggesting the occurrence of large groups of coregulated genes controlled by single markers.     

Arabidopsis PAI gene arrangements, cytosine methylation and expression.

Previous analysis of the PAI tryptophan biosynthetic gene family in Arabidopsis thaliana revealed that the Wassilewskija (WS) ecotype has four PAI genes at three unlinked sites: a tail-to-tail inverted repeat at one locus (PAI1-PAI4) plus singlet genes at two other loci (PAI2 and PAI3). The four WS PAI genes are densely cytosine methylated over their regions of DNA identity. In contrast, the Columbia (Col) ecotype has three singlet PAI genes at the analogous loci (PAI1, PAI2, and PAI3) and no cytosine methylation. To understand the mechanism of PAI gene duplication at the polymorphic PAI1 locus, and to investigate the relationship between PAI gene arrangement and PAI gene methylation, we analyzed 39 additional ecotypes of Arabidopsis. Six ecotypes had PAI arrangements similar to WS, with an inverted repeat and dense PAI methylation. All other ecotypes had PAI arrangements similar to Col, with no PAI methylation. The novel PAI-methylated ecotypes provide insights into the mechanisms underlying PAI gene duplication and methylation, as well as the relationship between methylation and gene expression.     

Mitochondrial DNA variations and nuclear RFLPs reflect different genetic similarities among 23 Arabidopsis thaliana ecotypes

The mitochondrial genome of 23 Arabidopsis thaliana ecotypes was analysed by Southern hybridization in total cellular DNA. Firstly, the extent of divergence between the mitochondrial genomes in closely related lines of one plant species and secondly, the use of mitochondrial versus nuclear RFLPs to determine evolutionary relationships between Arabidopsis ecotype isolates was investigated. Highly divergent stoichiometries of alternative mitochondrial genome arrangements characterize individual ecotypes including the complete loss of a 5 kb region from ecotype Landsberg without apparent effect on plant viability. The genetic similarities between ecotypes suggested by mitochondrial genome arrangements differ from those deduced from 18 nuclear RFLP loci (CAPS markers). Similarity of nuclear RFLP patterns among the 23 Arabidopsis ecotypes neitehr correlates with their geographic origin nor with the observed mitochondrial genome arrangements. A promiscuous mitochondrial sequence insertion previously identified in ecotype Columbia is also found in the nuclear genomes of ecotypes Eifel, Enkheim and Hilversum. Two ecotypes (Eifel and Tabor) displaying identical RFLP patterns at all 18 nuclear loci show differences in both this sequence transfer and a mitochondrial DNA recombination event.     

An allelic series for the chalcone synthase locus in Arabidopsis

Five new alleles of the Arabidopsis chalcone synthase (CHS) locus, tt4, have been characterized at the gene, protein, and end product levels as a genetic approach to understanding structure-function relationships in a key enzyme of plant secondary metabolism. Together with two previously described mutants, these tt4 lines represent one of the first allelic series for a central enzyme of the flavonoid pathway and include both null alleles and alleles with leaky, apparently temperature-sensitive, phenotypes. A variety of effects on accumulation of CHS protein and flavonoid glycosides were observed among these lines, including alterations in the apparent stability and activity of the enzyme. Assembly of the CHS homodimer also appeared to be impacted in several cases. A three-dimensional model of the Arabidopsis CHS protein, based on the recently determined structure for alfalfa CHS, predicts significant effects on protein structure or folding for several of the mutations. This allelic series should provide a useful genetic resource for ongoing studies of flavonoid enzyme structure, function, and subcellular organization.     

Recent stable insertion of mitochondrial DNA into an Arabidopsis polyubiquitin gene by nonhomologous recombination.

Sequence analysis of a newly identified polyubiquitin gene (UBQ13) from the Columbia ecotype of Arabidopsis thaliana revealed that the gene contained a 3.9-kb insertion in the coding region. All subclones of the 3.9-kb insert hybridized to isolated mitochondrial DNA. The insert was found to consist of at least two, possibly three, distinct DNA segments from the mitochondrial genome. A 590-bp region of the insert is nearly identical to the Arabidopsis mitochondrial nad1 gene. UBQ13 restriction fragments in total cellular DNA from ecotypes Ler, No-0, Be-0, WS, and RLD were identified and, with the exception of Be-0, their sizes were equivalent to that predicted from the corresponding ecotype Columbia UBQ13 restriction fragment without the mitochondrial insert. Isolation by polymerase chain reaction and sequence determination of UBQ13 sequences from the other ecotypes showed that all lacked the mitochondrial insert. All ecotypes examined, except Columbia, contain intact open reading frames in the region of the insert, including four ubiquitin codons which Columbia lacks. This indicates that the mitochondrial DNA in UBQ13 in ecotype Columbia is the result of an integration event that occurred after speciation of Arabidopsis rather than a deletion event that occurred in all ecotypes except Columbia. This stable movement of mitochondrial DNA to the nucleus is so recent that there are few nucleotide changes subsequent to the transfer event. This allows for precise analysis of the sequences involved and elucidation of the possible mechanism. The presence of intron sequences in the transferred nucleic acid indicates that DNA was the transfer intermediate. The lack of sequence identity between the integrating sequence and the target site, represented by the other Arabidopsis ecotypes, suggests that integration occurred via nonhomologus recombination. This nuclear/organellar gene transfer event is strikingly similar to the experimentally accessible process of nuclear integration of introduced heterologous DNA.     

The VLF loci, polymorphic between ecotypes Landsberg erecta and Columbia, dissect two branches of phytochrome A signal transduction that correspond to very-low-fluence and high-irradiance responses

Phytochromes play a key role in the perception of light signals by plants. In this study, the three classical phytochrome action modes, i.e. very-low-fluence responses (VLFR), low-fluence responses (LFR) and high-irradiance responses (HIR), were genetically dissected using phyA and phyB mutants of Arabidopsis thaliana (respectively lacking phytochrome A or phytochrome B) and a polymorphism between ecotypes Landsberg erecta and Columbia. Seed germination and potentiation of greening, hypocotyl growth inhibition and cotyledon unfolding in etiolated seedlings of the ecotype Landsberg erecta showed biphasic responses to the calculated proportion of active phytochrome established by one light pulse or repeated light pulses. The first phase, i.e. the VLFR, was absent in the phyA mutant, normal in the phyB mutant (both in the Landsberg erecta background) and severely deficient in Columbia. The second phase, i.e. the LFR, was present in the phyA mutant, deficient in the phyB mutant and normal in Columbia. Under continuous far-red light, HIR of etiolated seedlings were absent in phyA and normal in phyB and Columbia. The segregation of VLFR in recombinant inbred lines derived from a cross between Landsberg erecta and Columbia was analysed by MAPMAKER/QTL. Two quantitative trait loci, one on chromosome 2 ( VLF1 ) and another on chromosome 5 ( VLF2 ), were identified as responsible for the polymorphism. Phytochrome A is proposed to initiate two transduction pathways, VLFR and HIR, involving different cells and/or different molecular steps. This is the first application of the analysis of quantitative trait loci polymorphic between ecotypes to dissect transduction chains of environmental signals.     

RCH1, a locus in Arabidopsis that confers resistance to the hemibiotrophic fungal pathogen Colletotrichum higginsianum

When challenged with the crucifer pathogen Colletotrichum higginsianum, Arabidopsis thaliana ecotype Columbia (Col-0) was colonized by the fungus within 2 to 3 days, developing brown necrotic lesions surrounded by a yellow halo. Lesions spread from the inoculation site within 3 to 4 days, and subsequently continued to expand until they covered the entire leaf. Electron microscopy confirmed that C. higginsianum is a hemibiotroph on Arabidopsis, feeding initially on living cells as a biotroph before switching to a necrotrophic mode of growth. A collection of 37 ecotypes of Arabidopsis varied in their responses to infection by C. higginsianum. The ecotype Eil-0 was highly resistant, with symptoms limited to necrotic flecking and with only very limited fungal colonization. Analyses suggested that the hypersensitive response and reactive oxygen species may be important in this defense response. Expression analyses with cDNA microarrays indicated that the defense reaction depends primarily on the jasmonic acid- and ethylene-dependent signaling pathways and, to a lesser extent, on the salicylate-dependent pathway. Crosses between the Eil-0 and Col-0 ecotypes suggested that the resistance in Eil-0 was dominant and was conferred by a single locus, which we named RCH1. RCH1 is the first resistance locus to be identified from Arabidopsis against the hemibiotrophic fungus genus Colletotrichum.     

A new Ac-like transposon of Arabidopsis is associated with a deletion of the RPS5 disease resistance gene

The RPS5 and RFL1 disease resistance genes of Arabidopsis ecotype Col-0 are oriented in tandem and are separated by 1.4 kb. The Ler-0 ecotype contains RFL1, but lacks RPS5. Sequence analysis of the RPS5 deletion region in Ler-0 revealed the presence of an Ac-like transposable element, which we have designated Tag2. Southern hybridization analysis of six Arabidopsis ecotypes revealed 4-11 Tag2-homologous sequences in each, indicating that this element is ubiquitous in Arabidopsis and has been active in recent evolutionary time. The Tag2 insertion adjacent to RFL1 was unique to the Ler-0 ecotype, however, and was not present in two other ecotypes that lack RPS5. DNA sequence from the latter ecotypes lacked a transposon footprint, suggesting that insertion of Tag2 occurred after the initial deletion of RPS5. The deletion breakpoint contained a 192-bp insertion that displayed hallmarks of a nonhomologous DNA end-joining event. We conclude that loss of RPS5 was caused by a double-strand break and subsequent repair, and cannot be attributed to unequal crossing over between resistance gene homologs.     

Biochemical and genetic characterization of Arabidopsis flavanone 3beta-hydroxylase

Flavanone 3beta-hydroxylase (F3H; EC 1.14.11.9) is a 2-oxoglutarate dependent dioxygenase that catalyzes the synthesis of dihydrokaempferol, the common precursor for three major classes of 3-hydroxy flavonoids, the flavonols, anthocyanins, and proanthocyanidins. This enzyme also competes for flux into the 3-deoxy flavonoid branch pathway in some species. F3H genes are increasingly being used, often together with genes encoding other enzymes, to engineer flavonoid synthesis in microbes and plants. Although putative F3H genes have been cloned in a large number of plant species, only a handful have been functionally characterized. Here we describe the biochemical properties of the Arabidopsis thaliana F3H (AtF3H) enzyme and confirm the activities of gene products from four other plant species previously identified as having high homology to F3H. We have also investigated the surprising "leaky" phenotype of AtF3H mutant alleles, uncovering evidence that two related flavonoid enzymes, flavonol synthase (EC 1.14.11.23) and anthocyanidin synthase (EC 1.14.11.19), can partially compensate for F3H in vivo. These experiments further indicate that the absence of F3H in these lines enables the synthesis of uncommon 3-deoxy flavonoids in the Arabidopsis seed coat.     

Comparative analysis of expressed sequence tags in resistant and susceptible ecotypes of Arabidopsis thaliana infected with cucumber mosaic virus

Arabidopsis thaliana ecotype Columbia (Col-0) is susceptible to the yellow strain of cucumber mosaic virus [CMV(Y)], whereas ecotype C24 is resistant to CMV(Y). Comprehensive analyses of approximately 9,000 expressed sequence tags in ecotypes Col-0 and C24 infected with CMV(Y) suggested that the gene expression patterns in the two ecotypes differed. At 6, 12, 24 and 48 h after CMV(Y) inoculation, the expression of 6, 30, 85 and 788 genes, respectively, had changed in C24, as opposed to 20, 80, 53 and 150 genes in CMV(Y)-infected Col-0. At 12, 24 and 48 h after CMV(Y) inoculation, the abundance of 3, 10 and 55 mRNAs was altered in both ecotypes. However, at 6 h after CMV(Y) inoculation, no genes were co-induced or co-suppressed in both ecotypes. This differential pattern of gene expression between the two ecotypes at an early stage of CMV(Y) infection indicated that the cellular response for resistance may differ from that resulting in susceptibility at the level detectable by the macroarray. According to the expression pattern at various stages of infection, the expression of many genes could be grouped into clusters using cluster analysis. About 100 genes that encode proteins involved in chloroplast function were categorized into clusters 1 and 4, which had a differentially lower expression in CMV(Y)-inoculated C24. The expression of various genes encoding proteins in the endomembrane system belonged to clusters 2 and 4, which were induced in CMV(Y)-inoculated C24 and Col-0 leaves. Characterization of CMV(Y)-altered gene expression in the two ecotypes will contribute to a better understanding of the molecular basis of compatible and incompatible interactions between virus and host plants.     

Characterization of a new Arabidopsis mutant exhibiting enhanced disease resistance

In many plant-pathogen interactions, resistance is associated with the synthesis and accumulation of salicylic acid (SA) and pathogenesis-related (PR) proteins. At least two general classes of mutants with altered resistance to pathogen attack have been identified in Arabidopsis. One class exhibits increased susceptibility to pathogen infection; the other class exhibits enhanced resistance to pathogens. In an attempt to identify mutations in resistance-associated loci, we screened a population of T-DNA tagged Arabidopsis thaliana ecotype Wassilewskija (Ws) for mutants showing constitutive expression of the PR-1 gene (cep). A mutant was isolated and shown to constitutively express PR-1, PR-2, and PR-5 genes. This constitutive phenotype segregated as a single recessive trait in the Ws genetic background. The mutant also had elevated levels of SA, which are responsible for the cep phenotype. The cep mutant spontaneously formed hypersensitive response (HR)-like lesions on the leaves and cotyledons and also exhibited enhanced resistance to virulent bacterial and fungal pathogens. Genetic analyses of segregating progeny from outcrosses to other ecotypes unexpectedly revealed that alterations in more than one gene condition the constitutive expression of PR genes in the original mutant. One of the mutations, designated cpr20, maps to the lower arm of chromosome 4 and is required for the cep phenotype. Another mutation, which has been termed cpr21, maps to chromosome 1 and is often, but not always, associated with this phenotype. The recessive nature of the cep trait suggests that the CPR20 and CPR21 proteins may act as negative regulators in the disease resistance signal transduction pathway.     

Towards the cloning of GLY1

The Arabidopsis mutants designated gly1 exhibit a reduced carbon flux through the prokaryotic pathway that is compensated for by an increased carbon flux through the eukaryotic pathway. Biochemical approaches reveal that the gly1 phenotype cannot be explained by a deficiency in the enzymes of the prokaryotic pathway. The chemical complementation of the mutant phenotype by exogenous glycerol treatment of gly1 plants suggests a lesion affecting the glycerol 3-phosphate supply within the chloroplast. As an alternative to the biochemical study of the gly1 mutants we set out to map the GLY1 locus. The gly1 mutant being an EMS (ethyl methane sulphonate) mutant, we used a strategy based on the polymorphism existing between Arabidopsis ecotypes, here Columbia (gly1 background) and Landsberg erecta. We mapped gly1 on chromosome II. During the process of chromosome walking, the complete sequence of chromosome II was released, allowing us to make assumptions on candidate genes based on map location. We are currently sequencing the putative genes.     

A simple procedure for the analysis of single nucleotide polymorphisms facilitates map-based cloning in Arabidopsis

We developed a modified allele-specific PCR procedure for assaying single nucleotide polymorphisms (SNPs) and used the procedure (called SNAP for single-nucleotide amplified polymorphisms) to generate 62 Arabidopsis mapping markers. SNAP primers contain a single base pair mismatch within three nucleotides from the 3' end of one allele (the specific allele) and in addition have a 3' mismatch with the nonspecific allele. A computer program called SNAPER was used to facilitate the design of primers that generate at least a 1,000-fold difference in the quantity of the amplification products from the specific and nonspecific SNP alleles. Because SNAP markers can be readily assayed by electrophoresis on standard agarose gels and because a public database of over 25,000 SNPs is available between the Arabidopsis Columbia and Landsberg erecta ecotypes, the SNAP method greatly facilitates the map-based cloning of Arabidopsis genes defined by a mutant phenotype.     

Morphological and physiological traits of three major Arabidopsis thaliana accessions

Arabidopsis is currently the most studied organism in plant biology. Its short life cycle and small genome size have rendered it one of the principal model systems. Additionally, numerous large T-DNA insertion mutant collections are available. The advent of molecular biology and the completion of the Arabidopsis genome sequence have contributed to helping researchers discover a large variety of mutants identified for their phenotypes. Yet, it is important to consider that natural phenotypic variations exist and appear in natural ecotypes, differing greatly in several traits. Although there are a vast number of ecotypes available, only a few have been extensively studied, and some have been created in laboratories. In order to identify new phenotypic differences, we chose to study the differences observed between three ecotypes: Columbia (Col-0), Landsberg erecta (Laer-0) and Wassilewskija (Ws-0). Our research focuses on observable morphological traits throughout plant growth and development along the entire plant life cycle. We then attempted to shed some light on phenotypic discrepancies through the study of the class III peroxidase protein family, which is involved in many aspects of plant growth and tissue differentiation. Both morphological and molecular aspects reveal that there are major variations between ecotypes, hence indicating a possibly interesting heterotic effect in the F1 from crosses between different Arabidopsis ecotypes.