Tetraploid Arabidopsis Research Articles

Meiosis: Disentangling polyploid chromosomes with supercharged crossover interference

Meiosis depends on the cell's ability to match each chromosome to its homolog in a strictly pairwise fashion. A new study describes an elegant mechanism that tetraploid Arabidopsis arenosa plants evolved to faithfully connect and segregate pairs of homologous chromosomes.

Polyploidy-associated paramutation in Arabidopsis is determined by small RNAs, temperature, and allele structure.

Paramutation is a form of non-Mendelian inheritance in which the expression of a paramutable allele changes when it encounters a paramutagenic allele. This change in expression of the paramutable alleles is stably inherited even after segregation of both alleles. While the discovery of paramutation and studies of its underlying mechanism were made with alleles that change plant pigmentation, paramutation-like phenomena are known to modulate the expression of other traits and in other eukaryotes, and many cases have probably gone undetected. It is likely that epigenetic mechanisms are responsible for the phenomenon, as paramutation forms epialleles, genes with identical sequences but different expression states. This could account for the intergenerational inheritance of the paramutated allele, providing profound evidence that triggered epigenetic changes can be maintained over generations. Here, we use a case of paramutation that affects a transgenic selection reporter gene in tetraploid Arabidopsis thaliana. Our data suggest that different types of small RNA are derived from paramutable and paramutagenic epialleles. In addition, deletion of a repeat within the epiallele changes its paramutability. Further, the temperature during the growth of the epiallelic hybrids determines the degree and timing of the allelic interaction. The data further make it plausible why paramutation in this system becomes evident only in the segregating F2 population of tetraploid plants containing both epialleles. In summary, the results support a model for polyploidy-associated paramutation, with similarities as well as distinctions from other cases of paramutation.

Model-based genotype and ancestry estimation for potential hybrids with mixed-ploidy.

Non-random mating among individuals can lead to spatial clustering of genetically similar individuals and population stratification. This deviation from panmixia is commonly observed in natural populations. Consequently, individuals can have parentage in single populations or involving hybridization between differentiated populations. Accounting for this mixture and structure is important when mapping the genetics of traits and learning about the formative evolutionary processes that shape genetic variation among individuals and populations. Stratified genetic relatedness among individuals is commonly quantified using estimates of ancestry that are derived from a statistical model. Development of these models for polyploid and mixed-ploidy individuals and populations has lagged behind those for diploids. Here, we extend and test a hierarchical Bayesian model, called entropy, which can use low-depth sequence data to estimate genotype and ancestry parameters in autopolyploid and mixed-ploidy individuals (including sex chromosomes and autosomes within individuals). Our analysis of simulated data illustrated the trade-off between sequencing depth and genome coverage and found lower error associated with low-depth sequencing across a larger fraction of the genome than with high-depth sequencing across a smaller fraction of the genome. The model has high accuracy and sensitivity as verified with simulated data and through analysis of admixture among populations of diploid and tetraploid Arabidopsis arenosa.

The expansion of mesophyll cells is coordinated with the division of chloroplasts in diploid and tetraploid Arabidopsis thaliana.

Cell expression is coordinated with chloroplast division in diploid and tetraploid Arabidopsis thaliana, polyploidy promoted the expansion of mesophyll cells and chloroplast division in A. thaliana. Cell development and differentiation are always accompanied by cell expansion and chloroplast division in plants, but the relationship between them is still relatively unknown. To confirm the relationship between cell expansion and chloroplast division during the leaf development process of diploid and tetraploid Arabidopsis thaliana, we systematically analyzed the expansion of mesophyll cells and the division of chloroplasts through cytological observation and gene-expression characteristics. As a result, in diploid and tetraploid A. thaliana, there were two peaks in both mesophyll cell expansion and chloroplast division during the leaf development process. Tetraploid A. thaliana mesophyll cells were larger and contained more chloroplasts than diploid A. thaliana mesophyll cells, which indicated that cell division and cell expansion were coordinated with chloroplast division in A. thaliana and that polyploidy further promoted mesophyll cell expansion and chloroplast division.

Monitoring single-cell bioluminescence of Arabidopsis leaves to quantitatively evaluate the efficiency of a transiently introduced CRISPR/Cas9 system targeting the circadian clock gene ELF3.

The rapid assessment of gene function is crucial in biological research. The CRISPR/Cas9 system is widely used as a tool for targeted gene editing in many organisms including plants. Previously, we established a transient gene expression system for investigating cellular circadian rhythms in duckweed. In this system, circadian reporters and clock gene effectors-such as overexpressors, RNA interference (RNAi), and CRISPR/Cas9-were introduced into duckweed cells using a particle bombardment method. In the present study, we applied the CRISPR/Cas9 system at a single cell level to Arabidopsis thaliana, a model organism in plant biology. To evaluate the mutation induction efficiency of the system, we monitored single-cell bioluminescence after application of the CRISPR/Cas9 system targeting the ELF3 gene, which is essential for robust circadian rhythmicity. We evaluated the mutation induction efficiency by determining the proportion of cells with impaired circadian rhythms. Three single guide RNAs (sgRNAs) were designed, and the proportion of arrhythmic cells following their use ranged from 32 to 91%. A comparison of the mutation induction efficiencies of diploid and tetraploid Arabidopsis suggested that endoreduplication had a slight effect on efficiency. Taken together, our results demonstrate that the transiently introduced CRISPR/Cas9 system is useful for rapidly assessing the physiological function of target genes in Arabidopsis cells.

Genetic basis and evolution of rapid cycling in railway populations of tetraploid Arabidopsis arenosa.

Spatially structured plant populations with diverse adaptations provide powerful models to investigate evolution. Human-generated ruderal habitats are abundant and low-competition, but are challenging for plants not adapted to them. Ruderal habitats also sometimes form networked corridors (e.g. roadsides and railways) that allow rapid long-distance spread of successfully adapted variants. Here we use transcriptomic and genomic analyses, coupled with genetic mapping and transgenic follow-up, to understand the evolution of rapid cycling during adaptation to railway sites in autotetraploid Arabidopsis arenosa. We focus mostly on a hybrid population that is likely a secondary colonist of a railway site. These mountain railway plants are phenotypically similar to their cosmopolitan cousins. We thus hypothesized that colonization primarily involved the flow of adaptive alleles from the cosmopolitan railway variant. But our data shows that it is not that simple: while there is evidence of selection having acted on introgressed alleles, selection also acted on rare standing variation, and new mutations may also contribute. Among the genes we show have allelic divergence with functional relevance to flowering time are known regulators of flowering, including FLC and CONSTANS. Prior implications of these genes in weediness and rapid cycling supports the idea that these are “evolutionary hotspots” for these traits. We also find that one of two alleles of CONSTANS under selection in the secondary colonist was selected from rare standing variation in mountain populations, while the other was introgressed from the cosmopolitan railway populations. The latter allele likely arose in diploid populations over 700km away, highlighting how ruderal populations could act as allele conduits and thus influence local adaptation.

Deciphering the physiological and molecular mechanisms for copper tolerance in autotetraploid Arabidopsis.

Autotetraploid Arabidopsis line esd and 4COL exhibit enhanced tolerance to Cu stress by enhancing activation of antioxidative defenses, altering expression of genes related to Cu transport, chelation, and ABA-responsive. Autopolyploidy is ubiquitous among angiosperms and often results in better adaptation to stress conditions. Although copper (Cu) is an essential trace element, excess amounts can inhibit plant growth and even result in death. Here, we report that autotetraploid Arabidopsis thaliana esd and 4COL exhibit higher tolerance to Cu stress. Under such conditions, tetraploid plants had lower Cu contents and significantly more biomass compared with diploid plants. When exposed to excess Cu for 24h, levels of superoxide anions, hydrogen peroxide, and malondialdehyde were lower in tetraploids than in diploids. Moreover, activities of the antioxidant enzymes superoxide dismutase and peroxidase were stimulated and glutathione content was maintained at a relative higher level in the tetraploids. The expression of genes related to Cu transport and chelation was altered in autotetraploid Arabidopsis under Cu stress, and several key genes involved in the response to abscisic acid (ABA) were significantly up-regulated. Our results indicate that tetraploid Arabidopsis esd and 4COL acquire improved tolerance to Cu stress through enhanced activation of antioxidative defense mechanisms, altered expression of genes related to Cu transport and chelation, and positive regulation of expression for ABA-responsive genes.

Generation of stable nulliplex autopolyploid lines of Arabidopsis thaliana using CRISPR/Cas9 genome editing.

RNA-guided endonuclease-mediated targeted mutagenesis using the clustered regularly interspersed short palindromic repeats (CRISPR)/Cas9 system has been successful at targeting specific loci for modification in plants. While polyploidy is an evolutionary mechanism enabling plant adaptation, the analysis of gene function in polyploid plants has been limited due to challenges associated with generating polyploid knockout mutants for all gene copies in polyploid plant lines. This study investigated whether CRISPR/Cas9 mediated targeted mutagenesis can generate nulliplex tetraploid mutant lines in Arabidopsis thaliana, while also comparing the relative efficiency of targeted mutagenesis in tetraploid (4x) versus diploid (2x) backgrounds. Using CRISPR/Cas9 genome editing to generate knockout alleles of the TTG1 gene, we demonstrate that homozygous nulliplex mutants can be directly generated in tetraploid Arabidopsis thaliana plants. CRISPR/Cas9 genome editing now provides a route to more efficient generation of polyploid mutants for improving understanding of genome dosage effects in plants.

Intraspecific comparative analyses of metabolites between diploid and tetraploid Arabidopsis thaliana and Pyrus communis

BackgroundIt has been said that naturally occurring autopolyploid strains are more tolerant of biotic and/or abiotic stresses, due at least in part to the higher accumulation of secondary metabolites. Data supporting this hypothesis come from comparisons between naturally established autopolyploids and diploids; thus the high accumulation of metabolites in polyploid strains may be a secondarily acquired feature and not a direct effect of the autopolyploidy. But no detailed studies on this issue have been carried out. ResultsHere we carried out metabolome analyses between newly created tetraploids and the parent diploid in a model plant, Arabidopsis thaliana, and the agriculturally important pear fruit tree (Pyrus communis var. sativa). Our data showed that small numbers of metabolite species differ in amount between diploids and tetraploids in both species, but the differences were not reproducible among growth conditions and species. ConclusionsThese results strongly indicate that metabolite content is not universal nor the direct target of polyploidy-dependent changes. Instead, naturally occurring hyperaccumulation of metabolites in autopolyploids may be the result of secondary natural selection.

Selection on meiosis genes in diploid and tetraploid Arabidopsis arenosa.

Meiotic chromosome segregation is critical for fertility across eukaryotes, and core meiotic processes are well conserved even between kingdoms. Nevertheless, recent work in animals has shown that at least some meiosis genes are highly diverse or strongly differentiated among populations. What drives this remains largely unknown. We previously showed that autotetraploid Arabidopsis arenosa evolved stable meiosis, likely through reduced crossover rates, and that associated with this there is strong evidence for selection in a subset of meiosis genes known to affect axis formation, synapsis, and crossover frequency. Here, we use genome-wide data to study the molecular evolution of 70 meiosis genes in a much wider sample of A. arenosa. We sample the polyploid lineage, a diploid lineage from the Carpathian Mountains, and a more distantly related diploid lineage from the adjacent, but biogeographically distinct Pannonian Basin. We find that not only did selection act on meiosis genes in the polyploid lineage but also independently on a smaller subset of meiosis genes in Pannonian diploids. Functionally related genes are targeted by selection in these distinct contexts, and in two cases, independent sweeps occurred in the same loci. The tetraploid lineage has sustained selection on more genes, has more amino acid changes in each, and these more often affect conserved or potentially functional sites. We hypothesize that Pannonian diploid and tetraploid A. arenosa experienced selection on structural proteins that mediate sister chromatid cohesion, the formation of meiotic chromosome axes, and synapsis, likely for different underlying reasons.

Reference genes for normalizing transcription in diploid and tetraploid Arabidopsis.

Published transcription data from a set of 19 diploid Arabidopsis thaliana and 5 tetraploid (3 allo- and 2 auto- tetraploid) Arabidopsis accessions were re-analysed to identify reliable reference genes for normalization purposes. Five conventional and 16 novel reference genes previously derived from microarray data covering a wide range of abundance in absolute expression levels in diploid A. thaliana Col-0 were employed. Transcript abundance was well conserved for all 21 potential reference genes in the diploid A. thaliana accessions, with geNorm and NormFinder analysis indicating that AT5G46630, AT1G13320, AT4G26410, AT5G60390 and AT5G08290 were the most stable. However, conservation was less good among the tetraploid accessions, with the transcription of seven of the 21 genes being undetectable in all allotetraploids. The most stable gene was AT5G46630, while AT1G13440 was the unstable one. Hence, the choice of reference gene(s) for A. thaliana is quite wide, but with respect to the analysis of transcriptomic data derived from the tetraploids, it is probably necessary to select more than one reference gene.

Parameters Affecting Telomere-Mediated Chromosomal Truncation inArabidopsis

Conversion of a double-strand break into a telomere is a dangerous, potentially lethal event. However, little is known about the mechanism and control of de novo telomere formation (DNTF). DNTF can be instigated by the insertion of a telomere repeat array (TRA) into the host genome, which seeds the formation of a new telomere, resulting in chromosome truncation. Such events are rare and concentrated at chromosome ends. Here, we introduce tetraploid Arabidopsis thaliana as a robust genetic model for DNTF. Transformation of a 2.6-kb TRA into tetraploid plants resulted in a DNTF efficiency of 56%, fivefold higher than in diploid plants and 50-fold higher than in human cells. DNTF events were recovered across the entire genome, indicating that genetic redundancy facilitates recovery of DNTF events. Although TRAs as short as 100 bp seeded new telomeres, these tracts were unstable unless they were extended above a 1-kb size threshold. Unexpectedly, DNTF efficiency increased in plants lacking telomerase, and DNTF rates were lower in plants null for Ku70 or Lig4, components of the nonhomologous end-joining repair pathway. We conclude that multiple competing pathways modulate DNTF, and that tetraploid Arabidopsis will be a powerful model for elucidating the molecular details of these processes.

Polyploidization increases meiotic recombination frequency in Arabidopsis

BackgroundPolyploidization is the multiplication of the whole chromosome complement and has occurred frequently in vascular plants. Maintenance of stable polyploid state over generations requires special mechanisms to control pairing and distribution of more than two homologous chromosomes during meiosis. Since a minimal number of crossover events is essential for correct chromosome segregation, we investigated whether polyploidy has an influence on the frequency of meiotic recombination.ResultsUsing two genetically linked transgenes providing seed-specific fluorescence, we compared a high number of progeny from diploid and tetraploid Arabidopsis plants. We show that rates of meiotic recombination in reciprocal crosses of genetically identical diploid and autotetraploid Arabidopsis plants were significantly higher in tetraploids compared to diploids. Although male and female gametogenesis differ substantially in meiotic recombination frequency, both rates were equally increased in tetraploids. To investigate whether multivalent formation in autotetraploids was responsible for the increased recombination rates, we also performed corresponding experiments with allotetraploid plants showing strict bivalent pairing. We found similarly increased rates in auto- and allotetraploids, suggesting that the ploidy effect is independent of chromosome pairing configurations.ConclusionsThe evolutionary success of polyploid plants in nature and under domestication has been attributed to buffering of mutations and sub- and neo-functionalization of duplicated genes. Should the data described here be representative for polyploid plants, enhanced meiotic recombination, and the resulting rapid creation of genetic diversity, could have also contributed to their prevalence.

Haploid plants produced by centromere-mediated genome elimination

Production of haploid plants that inherit chromosomes from only one parent can greatly accelerate plant breeding. Haploids generated from a heterozygous individual and converted to diploid create instant homozygous lines, bypassing generations of inbreeding. Two methods are generally used to produce haploids. First, cultured gametophyte cells may be regenerated into haploid plants, but many species and genotypes are recalcitrant to this process. Second, haploids can be induced from rare interspecific crosses, in which one parental genome is eliminated after fertilization. The molecular basis for genome elimination is not understood, but one theory posits that centromeres from the two parent species interact unequally with the mitotic spindle, causing selective chromosome loss. Here we show that haploid Arabidopsis thaliana plants can be easily generated through seeds by manipulating a single centromere protein, the centromere-specific histone CENH3 (called CENP-A in human). When cenh3 null mutants expressing altered CENH3 proteins are crossed to wild type, chromosomes from the mutant are eliminated, producing haploid progeny. Haploids are spontaneously converted into fertile diploids through meiotic non-reduction, allowing their genotype to be perpetuated. Maternal and paternal haploids can be generated through reciprocal crosses. We have also exploited centromere-mediated genome elimination to convert a natural tetraploid Arabidopsis into a diploid, reducing its ploidy to simplify breeding. As CENH3 is universal in eukaryotes, our method may be extended to produce haploids in any plant species.

Cooperation of Multiple Chromatin Modifications Can Generate Unanticipated Stability of Epigenetic States inArabidopsis

Epigenetic changes of gene expression can potentially be reversed by developmental programs, genetic manipulation, or pharmacological interference. However, a case of transcriptional gene silencing, originally observed in tetraploid Arabidopsis thaliana plants, created an epiallele resistant to many mutations or inhibitor treatments that activate many other suppressed genes. This raised the question about the molecular basis of this extreme stability. A combination of forward and reverse genetics and drug application provides evidence for an epigenetic double lock that is only alleviated upon the simultaneous removal of both DNA methylation and histone methylation. Therefore, the cooperation of multiple chromatin modifications can generate unanticipated stability of epigenetic states and contributes to heritable diversity of gene expression patterns.

A large number of tetraploid Arabidopsis thaliana lines, generated by a rapid strategy, reveal high stability of neo-tetraploids during consecutive generations

Arabidopsis thaliana has, in conjunction with A. arenosa, developed into a system for the molecular analysis of alloplolyploidy. However, there are very few Arabidopsis lines available to study autopolyploidy. In order to investigate polyploidy on a reliable basis, we have optimised conventional methodologies and developed a novel strategy for the rapid generation and identification of polyploids based on trichome branching patterns. The analysis of more than two dozen independently induced Arabidopsis lines has led to interesting observations concerning the relationship between cell size and ploidy levels and on the relative stability of tetraploidy in Arabidopsis over at least three consecutive generations. The most important finding of this work is that neo-tetraploid lines exhibit considerable stability through all the generations tested. The systematic generation of tetraploid collections through this strategy as well as the lines generated in this work will help to unravel the consequences of polyploidy, particularly tetraploidy, on the genome, on gene expression and on natural diversity in Arabidopsis.

The allotetraploid Arabidopsis thaliana–Arabidopsis lyrata subsp. petraea as an alternative model system for the study of polyploidy in plants

Polyploidy is known to be common in plants and recent work has focused on the rapid changes in genome structure and expression that occur upon polyploidization. In Arabidopsis, much of this work has been done on a synthetic allotetraploid obtained by crossing a tetraploid Arabidopsis thaliana (2n = 4x = 20) with A. arenosa (2n = 4x = 32). To explore an alternative route to polyploidy in this model species, we have developed a synthetic allopolyploid by crossing two diploid species: A. thaliana (2n = 2x = 10) and Arabidopsis lyrata subsp. petraea (2n = 2x = 16). F(1) hybrids were easy to obtain and phenotypically more similar to A. lyrata. Spontaneous chromosome doubling events occurred in about 25% of the F(1)s, thus restoring fertility. The resulting allotetraploids (2n = 26) exhibited many genomic changes typically reported upon polyploidization. Nucleolar dominance was observed as only the A. lyrata rDNA loci were expressed in the F(1) and allotetraploids. Changes in the degree of methylation were observed at almost 25% of the loci examined by MSAP analysis. Finally, structural genomic alterations did occur as a large deletion covering a significant portion of the upper arm of chromosome II was detected but no evidence of increased mobility of transposons was obtained. Such allotetraploids derived from two parents with sequenced (or soon to be sequenced) genomes offer much promise in elucidating the various changes that occur in newly synthesized polyploids.

A mutation in the uvi4 gene promotes progression of endo‐reduplication and confers increased tolerance towards ultraviolet B light

We have isolated and characterized a new ultraviolet B (UV-B)-resistant mutant, uvi4 (UV-B-insensitive 4), of Arabidopsis. The fresh weight (FW) of uvi4 plants grown under supplemental UV-B light was more than twice that of the wild-type. No significant difference was found in their ability to repair the UV-B-induced cyclobutane pyrimidine dimers, or in the amount of UV-B absorptive compounds, both of which are well-known factors that contribute to UV sensitivity. Positional cloning revealed that the UVI4 gene encodes a novel basic protein of unknown function. We found that the hypocotyl cells in uvi4 undergo one extra round of endo-reduplication. The uvi4 mutation also promoted the progression of endo-reduplication during leaf development. The UVI4 gene is expressed mainly in actively dividing cells. In the leaves of P(UVI4)::GUS plants, the GUS signal disappeared in basipetal fashion as the leaf developed. The total leaf blade area was not different between uvi4 and the wild-type through leaf development, while the average cell area in the adaxial epidermis was considerably larger in uvi4, suggesting that the uvi4 leaves have fewer but larger epidermal cells. These results suggest that UVI4 is necessary for the maintenance of the mitotic state, and the loss of UVI4 function stimulated endo-reduplication. Tetraploid Arabidopsis was hyper-resistant to UV-B compared to diploid Arabidopsis, suggesting that the enhanced polyploidization is responsible for the increased UV-B tolerance of the uvi4 mutant.

Formation of stable epialleles and their paramutation-like interaction in tetraploid Arabidopsis thaliana.

Polyploidization is found frequently in plants, and species previously considered to be diploid may show remnants of earlier polyploidization events on closer inspection of their genomes. The success of polyploids may lie in increased genetic redundancy supporting subsequent genetic diversification. Although doubling the genome does not generate diversity per se, recent studies show that rapid genomic rearrangements and changes in DNA modification and gene expression patterns are associated with polyploid formation. But recessive modifications will not become phenotypically apparent in early polyploid generations. Here we show that epialleles in tetraploid plants (but not in diploids) interact in trans and lead to heritable gene silencing persisting after segregation from the inactivating allele. This mechanism, resembling paramutation, leads to the establishment of functional epigenetic homozygosity and, thus, to conversion of new recessive alleles into traits expressed in early polyploid generations. Such interactions probably contribute to rapid adaptation and evolution of polyploid plant species.

The basis of natural and artificial postzygotic hybridization barriers in Arabidopsis species.

The success or failure of interspecific crosses is vital to evolution and to agriculture, but much remains to be learned about the nature of hybridization barriers. Several mechanisms have been proposed to explain postzygotic barriers, including negative interactions between diverged sequences, global genome rearrangements, and widespread epigenetic reprogramming. Another explanation is imbalance of paternally and maternally imprinted genes in the endosperm. Interspecific crosses between diploid Arabidopsis thaliana as the seed parent and tetraploid Arabidopsis arenosa as the pollen parent produced seeds that aborted with the same paternal excess endosperm phenotype seen in crosses between diploid and hexaploid A. thaliana. Doubling maternal ploidy restored seed viability and normal endosperm morphology. However, substituting a hypomethylated tetraploid A. thaliana seed parent reestablished the hybridization barrier by causing seed abortion and a lethal paternal excess phenotype. We conclude from these findings that the dominant cause of seed abortion in the diploid A. thaliana x tetraploid A. arenosa cross is parental genomic imbalance. Our results also demonstrate that manipulation of DNA methylation can be sufficient to erect hybridization barriers, offering a potential mechanism for speciation and a means of controlling gene flow between species.