Thaliana Population Research Articles

NORE1/SAUL1 integrates temperature-dependent defense programs involving SGT1b and PAD4 pathways and leaf senescence in Arabidopsis.

Leaf senescence is not only primarily governed by developmental age but also influenced by various internal and external factors. Although some genes that control leaf senescence have been identified, the detailed regulatory mechanisms underlying integration of diverse senescence-associated signals into the senescence programs remain to be elucidated. To dissect the regulatory pathways involved in leaf senescence, we isolated the not oresara1-1 (nore1-1) mutant showing accelerated leaf senescence phenotypes from an EMS-mutagenized Arabidopsis thaliana population. We found that altered transcriptional programs in defense response-related processes were associated with the accelerated leaf senescence phenotypes observed in nore1-1 through microarray analysis. The nore1-1 mutation activated defense program, leading to enhanced disease resistance. Intriguingly, high ambient temperature effectively suppresses the early senescence and death phenotypes of nore1-1. The gene responsible for the phenotypes of nore1-1 contains a missense mutation in SENESCENCE-ASSOCIATED E3 UBIQUITIN LIGASE 1 (SAUL1), which was reported as a negative regulator of premature senescence in the light intensity- and PHYTOALEXIN DEFICIENT 4 (PAD4)-dependent manner. Through extensive double mutant analyses, we recently identified suppressor of the G2 Allele of SKP1b (SGT1b), one of the positive regulators for disease resistance conferred by many resistance (R) proteins, as a downstream signaling component in NORE1-mediated senescence and cell death pathways. In conclusion, NORE1/SAUL1 is a key factor integrating signals from temperature-dependent defense programs and leaf senescence in Arabidopsis. These findings provide a new insight that plants might utilize defense response program in regulating leaf senescence process, possibly through recruiting the related genes during the evolution of the leaf senescence program.

A Lipid Transfer Protein Increases the Glutathione Content and Enhances Arabidopsis Resistance to a Trichothecene Mycotoxin.

Fusarium head blight (FHB) or scab is one of the most important plant diseases worldwide, affecting wheat, barley and other small grains. Trichothecene mycotoxins such as deoxynivalenol (DON) accumulate in the grain, presenting a food safety risk and health hazard to humans and animals. Despite considerable breeding efforts, highly resistant wheat or barley cultivars are not available. We screened an activation tagged Arabidopsis thaliana population for resistance to trichothecin (Tcin), a type B trichothecene in the same class as DON. Here we show that one of the resistant lines identified, trichothecene resistant 1 (trr1) contains a T-DNA insertion upstream of two nonspecific lipid transfer protein (nsLTP) genes, AtLTP4.4 and AtLTP4.5. Expression of both nsLTP genes was induced in trr1 over 10-fold relative to wild type. Overexpression of AtLTP4.4 provided greater resistance to Tcin than AtLTP4.5 in Arabidopsis thaliana and in Saccharomyces cerevisiae relative to wild type or vector transformed lines, suggesting a conserved protection mechanism. Tcin treatment increased reactive oxygen species (ROS) production in Arabidopsis and ROS stain was associated with the chloroplast, the cell wall and the apoplast. ROS levels were attenuated in Arabidopsis and in yeast overexpressing AtLTP4.4 relative to the controls. Exogenous addition of glutathione and other antioxidants enhanced resistance of Arabidopsis to Tcin while the addition of buthionine sulfoximine, an inhibitor of glutathione synthesis, increased sensitivity, suggesting that resistance was mediated by glutathione. Total glutathione content was significantly higher in Arabidopsis and in yeast overexpressing AtLTP4.4 relative to the controls, highlighting the importance of AtLTP4.4 in maintaining the redox state. These results demonstrate that trichothecenes cause ROS accumulation and overexpression of AtLTP4.4 protects against trichothecene-induced oxidative stress by increasing the glutathione-based antioxidant defense.

Century-scale methylome stability in a recently diverged Arabidopsis thaliana lineage.

There has been much excitement about the possibility that exposure to specific environments can induce an ecological memory in the form of whole-sale, genome-wide epigenetic changes that are maintained over many generations. In the model plant Arabidopsis thaliana, numerous heritable DNA methylation differences have been identified in greenhouse-grown isogenic lines, but it remains unknown how natural, highly variable environments affect the rate and spectrum of such changes. Here we present detailed methylome analyses in a geographically dispersed A. thaliana population that constitutes a collection of near-isogenic lines, diverged for at least a century from a common ancestor. Methylome variation largely reflected genetic distance, and was in many aspects similar to that of lines raised in uniform conditions. Thus, even when plants are grown in varying and diverse natural sites, genome-wide epigenetic variation accumulates mostly in a clock-like manner, and epigenetic divergence thus parallels the pattern of genome-wide DNA sequence divergence.

A coastal cline in sodium accumulation in Arabidopsis thaliana is driven by natural variation of the sodium transporter AtHKT1;1.

The genetic model plant Arabidopsis thaliana, like many plant species, experiences a range of edaphic conditions across its natural habitat. Such heterogeneity may drive local adaptation, though the molecular genetic basis remains elusive. Here, we describe a study in which we used genome-wide association mapping, genetic complementation, and gene expression studies to identify cis-regulatory expression level polymorphisms at the AtHKT1;1 locus, encoding a known sodium (Na+) transporter, as being a major factor controlling natural variation in leaf Na+ accumulation capacity across the global A. thaliana population. A weak allele of AtHKT1;1 that drives elevated leaf Na+ in this population has been previously linked to elevated salinity tolerance. Inspection of the geographical distribution of this allele revealed its significant enrichment in populations associated with the coast and saline soils in Europe. The fixation of this weak AtHKT1;1 allele in these populations is genetic evidence supporting local adaptation to these potentially saline impacted environments.

Natural Diversity in Flowering Responses of Arabidopsis thaliana Caused by Variation in a Tandem Gene Array

Tandemly arrayed genes that belong to gene families characterize genomes of many organisms. Gene duplication and subsequent relaxation of selection can lead to the establishment of paralogous cluster members that may evolve along different trajectories. Here, we report on the structural variation in MADS AFFECTING FLOWERING 2 (MAF2) gene, one member of the tandemly duplicated cluster of MADS-box-containing transcription factors in Arabidopsis thaliana. The altered gene structure at the MAF2 locus is present as a moderate-frequency polymorphism in Arabidopsis and leads to the extensive diversity in transcript patterns due to alternative splicing. Rearrangements at the MAF2 locus are associated with an early flowering phenotype in BC(5) lines. The lack of suppression of flowering time in a MAF2-insertion line expressing the MAF2-specific artificial miRNA suggests that these MAF2 variants are behaving as loss-of-function alleles. The variation in gene architecture is also associated with segregation distortion, which may have facilitated the spread and the establishment of the corresponding alleles throughout the Eurasian range of the A. thaliana population.

Disruption of ptLPD1 or ptLPD2, genes that encode isoforms of the plastidial lipoamide dehydrogenase, confers arsenate hypersensitivity in Arabidopsis.

Arsenic is a ubiquitous environmental poison that inhibits root elongation and seed germination to a variable extent depending on the plant species. To understand the molecular mechanisms of arsenic resistance, a genetic screen was developed to isolate arsenate overly sensitive (aos) mutants from an activation-tagged Arabidopsis (Arabidopsis thaliana) population. Three aos mutants were isolated, and the phenotype of each was demonstrated to be due to an identical disruption of plastidial LIPOAMIDE DEHYDROGENASE1 (ptLPD1), a gene that encodes one of the two E3 isoforms found in the plastidial pyruvate dehydrogenase complex. In the presence of arsenate, ptlpd1-1 plants exhibited reduced root and shoot growth and enhanced anthocyanin accumulation compared with wild-type plants. The ptlpd1-1 plants accumulated the same amount of arsenic as wild-type plants, indicating that the aos phenotype was not due to increased arsenate in the tissues but to an increase in the innate sensitivity to the poison. Interestingly, a ptlpd1-4 knockdown allele produced a partial aos phenotype. Two loss-of-function alleles of ptLPD2 in Arabidopsis also caused elevated arsenate sensitivity, but the sensitivity was less pronounced than for the ptlpd1 mutants. Moreover, both the ptlpd1 and ptlpd2 mutants were more sensitive to arsenite than wild-type plants, and the LPD activity in isolated chloroplasts from wild-type plants was sensitive to arsenite but not arsenate. These findings show that the ptLPD isoforms are critical in vivo determinants of arsenite-mediated arsenic sensitivity in Arabidopsis and possible strategic targets for increasing arsenic tolerance.

A high-density collection of EMS-induced mutations for TILLING in Landsberg erecta genetic background of Arabidopsis

BackgroundArabidopsis thaliana is the main model species for plant molecular genetics studies and world-wide efforts are devoted to identify the function of all its genes. To this end, reverse genetics by TILLING (Targeting Induced Local Lesions IN Genomes) in a permanent collection of chemically induced mutants is providing a unique resource in Columbia genetic background. In this work, we aim to extend TILLING resources available in A. thaliana by developing a new population of ethyl methanesulphonate (EMS) induced mutants in the second commonest reference strain. In addition, we pursue to saturate the number of EMS induced mutations that can be tolerated by viable and fertile plants.ResultsBy mutagenizing with different EMS concentrations we have developed a permanent collection of 3712 M2/M3 independent mutant lines in the reference strain Landsberg erecta (Ler) of A. thaliana. This population has been named as the Arabidopsis TILLer collection. The frequency of mutations per line was maximized by using M1 plants with low but sufficient seed fertility. Application of TILLING to search for mutants in 14 genes identified 21 to 46 mutations per gene, which correspond to a total of 450 mutations. Missense mutations were found for all genes while truncations were selected for all except one. We estimated that, on average, these lines carry one mutation every 89 kb, Ler population providing a total of more than five million induced mutations. It is estimated that TILLer collection shows a two to three fold higher EMS mutation density per individual than previously reported A. thaliana population.ConclusionsAnalysis of TILLer collection demonstrates its usefulness for large scale TILLING reverse genetics in another reference genetic background of A. thaliana. Comparisons with TILLING populations in other organisms indicate that this new A. thaliana collection carries the highest chemically induced mutation density per individual known in diploid species.

Spatial distribution of leaf form and the self-thinning exponent are affected by the sensitivity of the response to abscisic acid in anArabidopsis thaliana population

The spatial distribution of leaves is related to the exponent of the self-thinning relationship in plant populations. In this study, we evaluated the fractal dimension of rosette leaves of wild-type (WT)Arabidopsis thaliana and of an abscisic acid (ABA) -insensitive mutant (abi2-1) to test a model of the spatial distribution of leaf form in anArabidopsis population based on subdivision of a cube surrounding the leaf into uniform boxes and to investigate ABA’s affect on this model of the leaf. The values of the self-thinning exponent were -1.31 and -1.45 for WT andabi2-1. The mean dimensions of the box used to model the spatial distribution of leaf form, estimated using our model, were 2.08 and 2.03, respectively. By assuming that the box dimension equals the fractal dimension within the populations, the predicted self-thinning exponent equaled -1.40 for WT and -1.49 forabi2-1. When exogenous ABA was applied to both genotypes, the self-thinning exponent became -1.26 and -1.43 for WT andabi2-1, and the exponents predicted using the dimensions of the box were -1.37 and -1.46, respectively. The empirically predicted exponent equaled that predicted using the dimensions of the box (95% confidence interval). Empirical prediction of the spatial pattern using the two genotypes with and without ABA showed that ABA influenced the spatial form of the rosette leaves. Therefore, sensitivity to ABA can affect self-thinning through genetically determined changes in leaf form and its spatial distribution.

Strong ecological but weak evolutionary effects of elevated CO2 on a recombinant inbred population of Arabidopsis thaliana

Increases in atmospheric CO2 concentration have an impact on plant communities by influencing plant growth and morphology, species interactions, and ecosystem processes. These ecological effects may be accompanied by evolutionary change if elevated CO2 (eCO2) alters patterns of natural selection or expression of genetic variation. Here, a statistically powerful quantitative genetic experiment and manipulations of CO2 concentrations in a field setting were used to investigate how eCO2 impacts patterns of selection on ecologically important traits in Arabidopsis thaliana; heritabilities, which influence the rate of response to selection; and genetic covariances between traits, which may constrain responses to selection. CO2 had strong phenotypic effects; plants grown in eCO2 were taller and produced more biomass and fruits. Also, significant directional selection was observed on many traits and significant genetic variation was observed for all traits. However, no evolutionary effect of eCO2 was detected; patterns of selection, heritabilities and genetic correlations corresponded closely in ambient and elevated CO2 environments. The data suggest that patterns of natural selection and the quantitative genetic parameters of this A. thaliana population are robust to increases in CO2 concentration and that responses to eCO2 will be primarily ecological.

Identification of an arsenic tolerant double mutant with a thiol-mediated component and increased arsenic tolerance in phyA mutants

A genetic screen was performed to isolate mutants showing increased arsenic tolerance using an Arabidopsis thaliana population of activation tagged lines. The most arsenic-resistant mutant shows increased arsenate and arsenite tolerance. Genetic analyses of the mutant indicate that the mutant contains two loci that contribute to arsenic tolerance, designated ars4 and ars5. The ars4ars5 double mutant contains a single T-DNA insertion, ars4, which co-segregates with arsenic tolerance and is inserted in the Phytochrome A (PHYA) gene, strongly reducing the expression of PHYA. When grown under far-red light conditions ars4ars5 shows the same elongated hypocotyl phenotype as the previously described strong phyA-211 allele. Three independent phyA alleles, ars4, phyA-211 and a new T-DNA insertion allele (phyA-t) show increased tolerance to arsenate, although to a lesser degree than the ars4ars5 double mutant. Analyses of the ars5 single mutant show that ars5 exhibits stronger arsenic tolerance than ars4, and that ars5 is not linked to ars4. Arsenic tolerance assays with phyB-9 and phot1/phot2 mutants show that these photoreceptor mutants do not exhibit phyA-like arsenic tolerance. Fluorescence HPLC analyses show that elevated levels of phytochelatins were not detected in ars4, ars5 or ars4ars5, however increases in the thiols cysteine, gamma-glutamylcysteine and glutathione were observed. Compared with wild type, the total thiol levels in ars4, ars5 and ars4ars5 mutants were increased up to 80% with combined buthionine sulfoximine and arsenic treatments, suggesting the enhancement of mechanisms that mediate thiol synthesis in the mutants. The presented findings show that PHYA negatively regulates a pathway conferring arsenic tolerance, and that an enhanced thiol synthesis mechanism contributes to the arsenic tolerance of ars4ars5.

Disruption of Arabidopsis thaliana MYB26 results in male sterility due to non-dehiscent anthers.

A male sterile mutant with a defect in anther dehiscence was identified in an Arabidopsis thaliana population mutagenized with the Zea mays transposon En-1/Spm. Mutants produce viable pollen that can fertilize when released mechanically from the anthers. Mutant stamens are of normal size and shape, but lack cell wall fortifications in the endothecial cell layer of the anther, which are required for the dehiscence process. The mutant phenotype was shown to be caused by a transposon insertion in AtMYB26, disrupting the putative DNA-binding domain of this R2R3-type MYB transcription factor. RT-PCR revealed that expression of AtMYB26 is restricted to inflorescences. Sterility was shown to be stable under several environmental conditions. The high stability of the sterile phenotype, together with the fact that pollen is functional, makes AtMYB26 and its orthologs a valuable tool for manipulating male fertility in higher plants.

Effects of genetic background on response to selection in experimental populations of Arabidopsis thaliana.

The extent to which genetic background can influence allelic fitness is poorly understood, despite having important evolutionary consequences. Using experimental populations of Arabidopsis thaliana and map-based population genetic data, we examined a multigeneration response to selection in populations with differentiated genetic backgrounds. Replicated experimental populations of A. thaliana with genetic backgrounds derived from ecotypes Landsberg and Niederzenz were subjected to strong viability and fertility selection by growing individuals from each population at high density for three generations in a growth chamber. Patterns of genome-wide selection were evaluated by examining deviations from expected frequencies of mapped molecular markers. Estimates of selection coefficients for individual genomic regions ranged from near 0 to 0.685. Genomic regions demonstrating the strongest response to selection most often were selected similarly in both genetic backgrounds. The selection response of several weakly selected regions, however, appeared to be sensitive to genetic background, but only one region showed evidence of positive selection in one background and negative selection in another. These results are most consistent with models of adaptive evolution in which allelic fitnesses are not strongly influenced by genetic background and only infrequently change in sign due to variation at other loci.

A. thaliana TRANSPARENT TESTA 1 is involved in seed coat development and defines the WIP subfamily of plant zinc finger proteins.

Seeds of the Arabidopsis thaliana transparent testa 1 mutant (tt1) appear yellow, due to the lack of condensed tannin pigments in the seed coat. The TT1 gene was isolated by reverse genetics using an En-1 transposon mutagenized A. thaliana population. TT1 gene expression was detected in developing ovules and young seeds only, and the gene was shown to encode a nuclear protein. Mutant seeds displayed altered morphology of the seed endothelium in which brown tannin pigments accumulate in wild-type plants, indicating that TT1 is involved in the differentiation of this cell layer. When overexpressed in transgenic A. thaliana plants, TT1 caused aberrant development and organ morphology. The protein contains a novel combination of two TFIIIA-type zinc finger motifs. Closely related motifs were detected in a number of putative proteins deduced from plant genomic and EST sequences. The new protein domain containing this type of zinc finger motifs was designated WIP, according to three strictly conserved amino acid residues. Our data indicate the existence of a small gene family in A. thaliana which is defined by the occurrence of the WIP domain. WIP genes may play important roles in regulating developmental processes, including the control of endothelium differentiation.

Successful PCR-based reverse genetic screens using an En-1-mutagenised Arabidopsis thaliana population generated via single-seed descent

The development of an Arabidopsis population via single-seed descent is described which includes 3,000 lines that carry approximately 15,000 independent insertions of the autonomous maize element En-1. A PCR strategy is outlined which allows the recovery of En-1-insertion mutants among this population in any random gene sequence of Arabidopsis thaliana. The method employs PCR reactions on pooled DNA. Positive amplification using a target-specific primer and an En-1-specific primer on row, column and single-tray pools identifies the putative insertion mutant. In a control experiment two insertion mutants of the PIN gene were successfully identified. In addition, a new independent insertion in the PIN gene was detected which was transmitted to the next generation and showed co-segregation with the pin phenotype. These examples demonstrate that the inheritance of inserts of the autonomous element En-1 is stable enough to make a proper genetic analysis feasible in a genomic background with multiple En-1 inserts.