The control of natural variation in cytosine methylation in Arabidopsis

The distance of pollen movement is an important determinant of the neighborhood area of plant populations. In earlier studies, we designed a method for estimating the distance of pollen dispersal, on the basis of the analysis of the differentiation among the pollen clouds of a sample of females, spaced across the landscape. The method was based solely on an estimate of the global level of differentiation among the pollen clouds of the total array of sampled females. Here, we develop novel estimators, on the basis of the divergence of pollen clouds for all pairs of females, assuming that an independent estimate of adult population density is available. A simulation study shows that the estimators are all slightly biased, but that most have enough precision to be useful, at least with adequate sample sizes. We show that one of the novel pairwise methods provides estimates that are slightly better than the best global estimate, especially when the markers used have low exclusion probability. The new method can also be generalized to the case where there is no prior information on the density of reproductive adults. In that case, we can jointly estimate the density itself and the pollen dispersal distance, given sufficient sample sizes. The bias of this last estimator is larger and the precision is lower than for those estimates based on independent estimates of density, but the estimate is of some interest, because a meaningful independent estimate of the density of reproducing individuals is difficult to obtain in most cases.     

Genetic mapping of natural variation in potassium concentrations in shoots of Arabidopsis thaliana

Naturally-occurring variation in K(+) concentrations between plant genotypes is potentially exploitable in a number of ways, including altering the relationship between K(+) accumulation and growth, enhancing salinity resistance, or improving forage quality. However, achieving these requires greater insight into the genetic basis of the variation in tissue K(+) concentrations. To this end, K(+) concentrations were measured in the shoots of 70 Arabidopsis thaliana accessions and a Cape Verdi Island/Landsberg erecta recombinant inbred line (RIL) population. The shoot K(+) concentrations expressed on the basis of fresh matter (KFM) or dry matter (KDM) were both broadly and normally distributed as was the shoot dry matter content per unit fresh weight (DMC). Using the data from the RILs, four quantitative trait loci (QTL) were identified for KFM and three for KDM. These were located on chromosomes 2, 3, 4, and 5. Two of the QTLs for KFM overlapped with those for KDM. None of these QTLs overlapped with those for fresh weight or dry weight, but the QTL for KDM located on chromosome 3 overlapped with one for DMC. In silico analysis was used to identify known or putative K(+) and cation transporter genes whose loci overlapped with the QTLs. In most cases, multiple genes were identified and the possible role of their gene products in determining shoot K(+) concentrations is discussed.     

Epigenetic natural variation in Arabidopsis thaliana

Cytosine methylation of repetitive sequences is widespread in plant genomes, occurring in both symmetric (CpG and CpNpG) as well as asymmetric sequence contexts. We used the methylation-dependent restriction enzyme McrBC to profile methylated DNA using tiling microarrays of Arabidopsis Chromosome 4 in two distinct ecotypes, Columbia and Landsberg erecta. We also used comparative genome hybridization to profile copy number polymorphisms. Repeated sequences and transposable elements (TEs), especially long terminal repeat retrotransposons, are densely methylated, but one third of genes also have low but detectable methylation in their transcribed regions. While TEs are almost always methylated, genic methylation is highly polymorphic, with half of all methylated genes being methylated in only one of the two ecotypes. A survey of loci in 96 Arabidopsis accessions revealed a similar degree of methylation polymorphism. Within-gene methylation is heritable, but is lost at a high frequency in segregating F₂ families. Promoter methylation is rare, and gene expression is not generally affected by differences in DNA methylation. Small interfering RNA are preferentially associated with methylated TEs, but not with methylated genes, indicating that most genic methylation is not guided by small interfering RNA. This may account for the instability of gene methylation, if occasional failure of maintenance methylation cannot be restored by other means.     

Complex genetics control natural variation in Arabidopsis thaliana resistance to Botrytis cinerea

The genetic architecture of plant defense against microbial pathogens may be influenced by pathogen lifestyle. While plant interactions with biotrophic pathogens are frequently controlled by the action of large-effect resistance genes that follow classic Mendelian inheritance, our study suggests that plant defense against the necrotrophic pathogen Botrytis cinerea is primarily quantitative and genetically complex. Few studies of quantitative resistance to necrotrophic pathogens have used large plant mapping populations to dissect the genetic structure of resistance. Using a large structured mapping population of Arabidopsis thaliana, we identified quantitative trait loci influencing plant response to B. cinerea, measured as expansion of necrotic lesions on leaves and accumulation of the antimicrobial compound camalexin. Testing multiple B. cinerea isolates, we identified 23 separate QTL in this population, ranging in isolate-specificity from being identified with a single isolate to controlling resistance against all isolates tested. We identified a set of QTL controlling accumulation of camalexin in response to pathogen infection that largely colocalized with lesion QTL. The identified resistance QTL appear to function in epistatic networks involving three or more loci. Detection of multilocus connections suggests that natural variation in specific signaling or response networks may control A. thaliana-B. cinerea interaction in this population.     

Novel loci control variation in reproductive timing in Arabidopsis thaliana in natural environments

Molecular biologists are rapidly characterizing the genetic basis of flowering in model species such as Arabidopsis thaliana. However, it is not clear how the developmental pathways identified in controlled environments contribute to variation in reproductive timing in natural ecological settings. Here we report the first study of quantitative trait loci (QTL) for date of bolting (the transition from vegetative to reproductive growth) in A. thaliana in natural seasonal field environments and compare the results with those obtained under typical growth-chamber conditions. Two QTL specific to long days in the chamber were expressed only in spring-germinating cohorts in the field, and two loci specific to short days in the chamber were expressed only in fall-germinating cohorts, suggesting differential involvement of the photoperiod pathway in different seasonal environments. However, several other photoperiod-specific QTL with large effects in controlled conditions were undetectable in natural environments, indicating that expression of allelic variation at these loci was overridden by environmental factors specific to the field. Moreover, a substantial number of QTL with major effects on bolting date in one or more field environments were undetectable under controlled environment conditions. These novel loci suggest the involvement of additional genes in the transition to flowering under ecologically relevant conditions.     

Variation of glucosinolate accumulation among different organs and developmental stages of Arabidopsis thaliana

The glucosinolate content of various organs of the model plant Arabidopsis thaliana (L.) Heynh., Columbia (Col-0) ecotype, was analyzed at different stages during its life cycle. Significant differences were noted among organs in both glucosinolate concentration and composition. Dormant and germinating seeds had the highest concentration (2.5-3.3% by dry weight), followed by inflorescences, siliques (fruits), leaves and roots. While aliphatic glucosinolates predominated in most organs, indole glucosinolates made up nearly half of the total composition in roots and late-stage rosette leaves. Seeds had a very distinctive glucosinolate composition. They possessed much higher concentrations of several types of aliphatic glucosinolates than other organs, including methylthioalkyl and, hydroxyalkyl glucosinolates and compounds with benzoate esters than other organs. From a developmental perspective, older leaves had lower glucosinolate concentrations than younger leaves, but this was not due to decreasing concentrations in individual leaves with age (glucosinolate concentration was stable during leaf expansion). Rather, leaves initiated earlier in development simply had much lower rates of glucosinolate accumulation per dry weight gain throughout their lifetimes. During seed germination and leaf senescence, there were significant declines in glucosinolate concentration. The physiological and ecological significance of these findings is briefly discussed.     

Natural variation in MAM within and between populations of Arabidopsis lyrata determines glucosinolate phenotype

The genetic variation that underlies the glucosinolate phenotype of Arabidopsis lyrata ssp. petraea was investigated between and within populations. A candidate glucosinolate biosynthetic locus (MAM, containing methylthioalkylmalate synthase genes) was mapped in A. lyrata to a location on linkage group 6 corresponding to the homologous location for MAM in A. thaliana. In A. thaliana MAM is responsible for side chain elongation in aliphatic glucosinolates, and the MAM phenotype can be characterized by the ratios of long- to short-chain glucosinolates. A quantitative trait loci (QTL) analysis of glucosinolate ratios in an A. lyrata interpopulation cross found one QTL at MAM. Additional QTL were identified for total indolic glucosinolates and for the ratio of aliphatic to indolic glucosinolates. MAM was then used as the candidate gene for a within-population cosegregation analysis in a natural A. lyrata population from Germany. Extensive variation in microsatellite markers at MAM was found and this variation cosegregated with the same glucosinolate ratios as in the QTL study. The combined results indicate that both between- and within-population genetic variation in the MAM region determines phenotypic variation in glucosinolate side chains in A. lyrata.     

Small RNA-directed epigenetic natural variation in Arabidopsis thaliana

Progress in epigenetics has revealed mechanisms that can heritably regulate gene function independent of genetic alterations. Nevertheless, little is known about the role of epigenetics in evolution. This is due in part to scant data on epigenetic variation among natural populations. In plants, small interfering RNA (siRNA) is involved in both the initiation and maintenance of gene silencing by directing DNA methylation and/or histone methylation. Here, we report that, in the model plant Arabidopsis thaliana, a cluster of approximately 24 nt siRNAs found at high levels in the ecotype Landsberg erecta (Ler) could direct DNA methylation and heterochromatinization at a hAT element adjacent to the promoter of FLOWERING LOCUS C (FLC), a major repressor of flowering, whereas the same hAT element in ecotype Columbia (Col) with almost identical DNA sequence, generates a set of low abundance siRNAs that do not direct these activities. We have called this hAT element MPF for Methylated region near Promoter of FLC, although de novo methylation triggered by an inverted repeat transgene at this region in Col does not alter its FLC expression. DNA methylation of the Ler allele MPF is dependent on genes in known silencing pathways, and such methylation is transmissible to Col by genetic crosses, although with varying degrees of penetrance. A genome-wide comparison of Ler and Col small RNAs identified at least 68 loci matched by a significant level of approximately 24 nt siRNAs present specifically in Ler but not Col, where nearly half of the loci are related to repeat or TE sequences. Methylation analysis revealed that 88% of the examined loci (37 out of 42) were specifically methylated in Ler but not Col, suggesting that small RNA can direct epigenetic differences between two closely related Arabidopsis ecotypes.     

Brassinosteroids regulate glucosinolate biosynthesis in Arabidopsis thaliana

Plants must constantly adjust their growth and defense responses to deal with the wide variety of stresses they encounter in their environment. Among phytohormones, brassinosteroids (BRs) are an important group of plant steroid hormones involved in numerous aspects of the plant lifecycle including growth, development and responses to various stresses including insect attacks. Here, we show that BRs regulate glucosinolate (GS) biosynthesis and function in insect herbivory. Preference tests and larval feeding experiments using the generalist herbivore, diamondback moth (Plutella xylostella), revealed that the larvae prefer to feed on Arabidopsis thaliana brassinosteroid insensitive 1 (bri1‐5) plants over wild‐type Ws‐2 or BRI1‐Flag (bri1‐5 background) transgenic plants, which results in an increase in larval weight. Analysis of GS contents showed that 3‐(methylsulfinyl) propyl GS (C3) levels were higher in bri1‐5 than in Ws2 and BRI1‐Flag transgenic plants, whereas sinigrin (2‐propenylglucosinolate), glucoerucin (4‐methylthiobutylglucosinolate) and glucobrassicin (indol‐3‐ylmethylglucosinolate) levels were lower in this mutant. We investigated the effect of brassinolide (BL) on GS biosynthesis in Arabidopsis and radish (Raphanus sativus L.) by monitoring the expression levels of GS biosynthetic genes, including MAM1, MAM3, BCAT4 and AOP2, which increased in a BL‐dependent manner. These results suggest that BRs regulate GS profiles in higher plants, which function in defense responses against insects.     

Proteomic investigation of natural variation between Arabidopsis ecotypes

Two-dimensional (2-D) gel electrophoresis and peptide mass fingerprinting were used to investigate the natural variation in the proteome among 8 Arabidopsis thaliana ecotypes, of which 3 were previously shown to display atypical responses to environmental stress. Comparison of 2-D maps demonstrated that only one-quarter of spots was shared by all accessions. On the other hand, only 15% of the 25 majors spots accounting for half the total protein amount could be classified as major spots in all ecotypes. Identification of these major spots demonstrated large differences between the major functions detected. Accordingly, the proteomes appeared to reveal important variations in terms of function between ecotypes. Hierarchical clustering of proteomes according to either the amount of all anonymous spots, that of the 25 major spots or the functions of these major spots identified the same classes of ecotypes, and grouped the three atypical ecotypes. It is proposed that proteome comparison has the capacity to evidence differences in the physiological status of ecotypes. Results are discussed with respect to the possibility to infer such differences from limited comparisons of major proteins. It is concluded that classical proteomics could constitute a powerful tool to mine the biodiversity between ecotypes of a single plant species.     

Major signaling pathways modulate Arabidopsis glucosinolate accumulation and response to both phloem-feeding and chewing insects

Plant responses to enemies are coordinated by several interacting signaling systems. Molecular and genetic studies with mutants and exogenous signal application suggest that jasmonate (JA)-, salicylate (SA)-, and ethylene (ET)-mediated pathways modulate expression of portions of the defense phenotype in Arabidopsis (Arabidopsis thaliana), but have not yet linked these observations directly with plant responses to insect attack. We compared the glucosinolate (GS) profiles of rosette leaves of 4-week-old mutant and transgenic Arabidopsis (Columbia) plants compromised in these three major signaling pathways, and characterized responses by those plants to feeding by two phloem-feeding aphids (generalist Myzus persicae and specialist Brevicoryne brassicae) and one generalist caterpillar species (Spodoptera exigua Hubner). Blocked JA signaling in coronatine-insensitive (coi1) and enhanced expression of SA-signaled disease resistance in hypersensitive response-like (hrl1) mutants reduced constitutive GS concentrations, while blocking SA signaling at the mediator protein npr1 mutant (NPR) increased them. There was no significant impact on constitutive GS contents of blocking ET signaling (at ET resistant [etr1]) or reducing SA concentrations (nahG transgene). We found increased GS accumulation in response to insect feeding, which required functional NPR1 and ETR1 but not COI1 or SA. Insect feeding caused increases primarily in short-chain aliphatic methylsulfinyl GS. By contrast, responses to exogenous JA, a frequent experimental surrogate for insect attack, were characterized by an increase in indolyl GS. Insect performance, measured as population increase or weight increase, was negatively related to GS levels, but we found evidence that other, ET-regulated factors may also be influential. Plant resistance to (consumption by) S. exigua was not related to insect growth because some plant chemistries inhibited growth while others inhibited feeding. These major signaling pathways modulate Arabidopsis GS accumulation and response to both phloem-feeding and chewing insects, often antagonistically; NPR appears to be central to these interactions. Our results indicate that exogenous signal application and plant consumption measures may not provide useful measures of plant responses to actual insect feeding.     

Genomic approaches to analyzing natural variation in Arabidopsis thaliana

The genomics tools available for studying Arabidopsis thaliana are a great resource for researchers trying to characterize and understand the genetic basis of natural variation. Abundant polymorphic markers aid quantitative trait locus (QTL) mapping, the fully sequenced genome provides rapid identification of candidate loci, and extensive knockout collections allow those candidate loci to be tested. Combining QTL mapping of classic phenotypic traits with biochemical or expression analysis is providing mechanistic insight into the traits of interest. Conversely, natural variation studies are now being done on genomic traits such as methylation or chiasma frequency.     

Genetic variation in Arabidopsis thaliana for night-time leaf conductance

Night-time leaf conductance ( g _night) and transpiration may have several adaptive benefits related to plant water, nutrient and carbon relations. Little is known, however, about genetic variation in g _night and whether this variation correlates with other gas exchange traits related to water use and/or native habitat climate. We investigated g _night in 12 natural accessions and three near isogenic lines (NILs) of Arabidopsis thaliana . Genetic variation in g _night was found for the natural accessions, and g _night was negatively correlated with native habitat atmospheric vapour pressure deficit (VPD_air), suggesting lower g _night may be favoured by natural selection in drier habitats. However, there were also significant genetic correlations of g _night with daytime gas exchange traits expected to affect plant fitness [i.e. daytime leaf conductance, photosynthesis and intrinsic water-use efficiency (WUE_i)], indicating that selection on daytime gas exchange traits may result in indirect selection on g _night. The comparison of three NILs to their parental genotypes identified one quantitative trait locus (QTL) contributing to variation in g _night. Further characterization of genetic variation in g _night within and among populations and species, and of associations with other traits and native habitats will be needed to understand g _night as a putatively adaptive trait.     

FRIGIDA-independent variation in flowering time of natural Arabidopsis thaliana accessions

FRIGIDA (FRI) and FLOWERING LOCUS C (FLC) are two genes that, unless plants are vernalized, greatly delay flowering time in Arabidopsis thaliana. Natural loss-of-function mutations in FRI cause the early flowering growth habits of many A. thaliana accessions. To quantify the variation among wild accessions due to FRI, and to identify additional genetic loci in wild accessions that influence flowering time, we surveyed the flowering times of 145 accessions in long-day photoperiods, with and without a 30-day vernalization treatment, and genotyped them for two common natural lesions in FRI. FRI is disrupted in at least 84 of the accessions, accounting for only approximately 40% of the flowering-time variation in long days. During efforts to dissect the causes for variation that are independent of known dysfunctional FRI alleles, we found new loss-of-function alleles in FLC, as well as late-flowering alleles that do not map to FRI or FLC. An FLC nonsense mutation was found in the early flowering Van-0 accession, which has otherwise functional FRI. In contrast, Lz-0 flowers late because of high levels of FLC expression, even though it has a deletion in FRI. Finally, eXtreme array mapping identified genomic regions linked to the vernalization-independent, late-flowering habit of Bur-0, which has an alternatively spliced FLC allele that behaves as a null allele.     

Looking for natural variation in chiasma frequency in Arabidopsis thaliana

Information concerning natural variation either in chiasma frequency or in the genetic basis of any such variation is a valuable tool to characterize phenotypic traits and their genetic control. Here meiotic recombination frequencies are analysed in nine geographically and ecologically diverse accessions of Arabidopsis thaliana, and a comparative study was carried out incorporating previous data from another eight accessions. Chiasma frequencies, estimated by counting rod and ring bivalents at metaphase I, varied up to 22% among accessions. However, no differences were found among plants of the same accession. There was a relationship, which does not necessarily imply direct proportionality, between the size of the chromosomes and their mean chiasma frequency. Chiasma frequency and distribution between arms and among chromosomes were not consistent over accessions. These findings indicate the existence of genetic factors controlling meiotic recombination both throughout the whole genome and at the whole chromosome level. The reliability of chiasma scoring as an indicator of reciprocal recombination events is also discussed.