Sureshkumar Balasubramanian

Diversity of flowering responses in wild Arabidopsis thaliana strains.

Although multiple environmental cues regulate the transition to flowering in Arabidopsis thaliana, previous studies have suggested that wild A. thaliana accessions fall primarily into two classes, distinguished by their requirement for vernalization (extended winter-like temperatures), which enables rapid flowering under long days. Much of the difference in vernalization response is apparently due to variation at two epistatically acting loci, FRI and FLC. We present the response of over 150 wild accessions to three different environmental variables. In long days, FLC is among those genes whose expression is most highly correlated with flowering. In short days, FRI and FLC are less important, although their contribution is still significant. In addition, there is considerable variation not only in vernalization response, but also in the response to differences in day length or ambient growth temperature. The identification of accessions that flower relatively early or late in specific environments suggests that many of the flowering-time pathways identified by mutagenesis, such as those that respond to day length, contribute to flowering-time variation in the wild. In contrast to differences in vernalization requirement, which are mainly mediated by FRI and FLC, it seems that variation in these other pathways is due to allelic effects at several different loci.

Natural allelic variation underlying a major fitness trade-off in Arabidopsis thaliana.

Plants can defend themselves against a wide array of enemies, from microbes to large animals, yet there is great variability in the effectiveness of such defences, both within and between species. Some of this variation can be explained by conflicting pressures from pathogens with different modes of attack. A second explanation comes from an evolutionary ‘tug of war’, in which pathogens adapt to evade detection, until the plant has evolved new recognition capabilities for pathogen invasion. If selection is, however, sufficiently strong, susceptible hosts should remain rare. That this is not the case is best explained by costs incurred from constitutive defences in a pest-free environment. Using a combination of forward genetics and genome-wide association analyses, we demonstrate that allelic diversity at a single locus, ACCELERATED CELL DEATH 6 (ACD6), underpins marked pleiotropic differences in both vegetative growth and resistance to microbial infection and herbivory among natural Arabidopsis thaliana strains. A hyperactive ACD6 allele, compared to the reference allele, strongly enhances resistance to a broad range of pathogens from different phyla, but at the same time slows the production of new leaves and greatly reduces the biomass of mature leaves. This allele segregates at intermediate frequency both throughout the worldwide range of A. thaliana and within local populations, consistent with this allele providing substantial fitness benefits despite its marked impact on growth.

The PHYTOCHROME C photoreceptor gene mediates natural variation in flowering and growth responses of Arabidopsis thaliana.

Light has an important role in modulating seedling growth and flowering time. We show that allelic variation at the PHYTOCHROME C (PHYC) photoreceptor locus affects both traits in natural populations of A. thaliana. Two functionally distinct PHYC haplotype groups are distributed in a latitudinal cline dependent on FRIGIDA, a locus that together with FLOWERING LOCUS C explains a large portion of the variation in A. thaliana flowering time. In a genome-wide scan for association of 65 loci with latitude, there was an excess of significant P values, indicative of population structure. Nevertheless, PHYC was the most strongly associated locus across 163 strains, suggesting that PHYC alleles are under diversifying selection in A. thaliana. Our work, together with previous findings, suggests that photoreceptor genes are major agents of natural variation in plant flowering and growth response.

Cis -regulatory Changes at FLOWERING LOCUS T Mediate Natural Variation in Flowering Responses of Arabidopsis thaliana

Flowering time, a critical adaptive trait, is modulated by several environmental cues. These external signals converge on a small set of genes that in turn mediate the flowering response. Mutant analysis and subsequent molecular studies have revealed that one of these integrator genes, FLOWERING LOCUS T (FT), responds to photoperiod and temperature cues, two environmental parameters that greatly influence flowering time. As the central player in the transition to flowering, the protein coding sequence of FT and its function are highly conserved across species. Using QTL mapping with a new advanced intercross-recombinant inbred line (AI-RIL) population, we show that a QTL tightly linked to FT contributes to natural variation in the flowering response to the combined effects of photoperiod and ambient temperature. Using heterogeneous inbred families (HIF) and introgression lines, we fine map the QTL to a 6.7 kb fragment in the FT promoter. We confirm by quantitative complementation that FT has differential activity in the two parental strains. Further support for FT underlying the QTL comes from a new approach, quantitative knockdown with artificial microRNAs (amiRNAs). Consistent with the causal sequence polymorphism being in the promoter, we find that the QTL affects FT expression. Taken together, these results indicate that allelic variation at pathway integrator genes such as FT can underlie phenotypic variability and that this may be achieved through cis-regulatory changes.

QTL mapping in new Arabidopsis thaliana advanced intercross-recombinant inbred lines

Background Even when phenotypic differences are large between natural or domesticated strains, the underlying genetic basis is often complex, and causal genomic regions need to be identified by quantitative trait locus (QTL) mapping. Unfortunately, QTL positions typically have large confidence intervals, which can, for example, lead to one QTL being masked by another, when two closely linked loci are detected as a single QTL. One strategy to increase the power of precisely localizing small effect QTL, is the use of an intercross approach before inbreeding to produce Advanced Intercross RILs (AI-RILs). Methodology/Principal Findings We present two new AI-RIL populations of Arabidopsis thaliana genotyped with an average intermarker distance of 600 kb. The advanced intercrossing design led to expansion of the genetic map in the two populations, which contain recombination events corresponding to 50 kb/cM in an F2 population. We used the AI-RILs to map QTL for light response and flowering time, and to identify segregation distortion in one of the AI-RIL populations due to a negative epistatic interaction between two genomic regions. Conclusions/Significance The two new AI-RIL populations, EstC and KendC, derived from crosses of Columbia (Col) to Estland (Est-1) and Kendallville (Kend-L) provide an excellent resource for high precision QTL mapping. Moreover, because they have been genotyped with over 100 common markers, they are also excellent material for comparative QTL mapping.

Control of lateral organ development and flowering time by the Arabidopsis thaliana MADS-box Gene AGAMOUS-LIKE6

MADS-domain transcription factors play pivotal roles in various developmental processes. The lack of simple loss-of-function phenotypes provides impediments to understand the biological function of some of the MADS-box transcription factors. Here we have characterized the potential role of the Arabidopsis thaliana AGAMOUS-LIKE6 (AGL6) gene by fusing full-length coding sequence with transcriptional activator and repressor domains and suggest a role for AGL6 in lateral organ development and flowering. Upon photoperiodic induction of flowering, AGL6 becomes expressed in abaxial and proximal regions of cauline leaf primordia, as well as the cryptic bracts subtending flowers. In developing flowers, AGL6 is detected in the proximal regions of all floral organs and in developing ovules. Converting AGL6 into a strong activator through fusion to the VP16 domain triggers bract outgrowth, implicating AGL6 in the development of bractless flowers in Arabidopsis. In addition, ectopic reproductive structures form on both bracts and flowers in gAGL6::VP16 transgenic plants, which is dependent on B and C class homeotic genes, but independent of LEAFY. Overexpression of both AGL6 and its transcriptional repressor form, AGL6::EAR, causes precocious flowering and terminal flower formation, suggesting that AGL6 suppresses the function of a floral repressor.

A Genetic Defect Caused by a Triplet Repeat Expansion in Arabidopsis thaliana

Variation in the length of simple DNA triplet repeats has been linked to phenotypic variability in microbes and to several human disorders. Population-level forces driving triplet repeat contraction and expansion in multicellular organisms are, however, not well understood. We have identified a triplet repeat–associated genetic defect in an Arabidopsis thaliana variety collected from the wild. The Bur-0 strain carries a dramatically expanded TTC/GAA repeat in the intron of the ISOPROPYL MALATE ISOMERASE LARGE SUB UNIT1 (IIL1; At4g13430) gene. The repeat expansion causes an environment-dependent reduction in IIL1 activity and severely impairs growth of this strain, whereas contraction of the expanded repeat can reverse the detrimental phenotype. The Bur-0 IIL1 defect thus presents a genetically tractable model for triplet repeat expansions and their variability in natural populations.

Inferring short tandem repeat variation from paired-end short reads

The advances of high-throughput sequencing offer an unprecedented opportunity to study genetic variation. This is challenged by the difficulty of resolving variant calls in repetitive DNA regions. We present a Bayesian method to estimate repeat-length variation from paired-end sequence read data. The method makes variant calls based on deviations in sequence fragment sizes, allowing the analysis of repeats at lengths of relevance to a range of phenotypes. We demonstrate the method’s ability to detect and quantify changes in repeat lengths from short read genomic sequence data across genotypes. We use the method to estimate repeat variation among 12 strains of Arabidopsis thaliana and demonstrate experimentally that our method compares favourably against existing methods. Using this method, we have identified all repeats across the genome, which are likely to be polymorphic. In addition, our predicted polymorphic repeats also included the only known repeat expansion in A. thaliana, suggesting an ability to discover potential unstable repeats.

Natural Variation in Biogenesis Efficiency of Individual Arabidopsis thaliana MicroRNAs

Like protein-coding genes, loci that produce microRNAs (miRNAs) are generally considered to be under purifying selection, consistent with miRNA polymorphisms being able to cause disease. Nevertheless, it has been hypothesized that variation in miRNA genes may contribute to phenotypic diversity. Here we demonstrate that a naturally occurring polymorphism in the MIR164A gene affects leaf shape and shoot architecture in Arabidopsis thaliana, with the effects being modified by additional loci in the genome. A single base pair substitution in the miRNA complementary sequence alters the predicted stability of the miRNA:miRNA(∗) duplex. It thereby greatly reduces miRNA accumulation, probably because it interferes with precursor processing. We demonstrate that this is not a rare exception and that natural strains of Arabidopsis thaliana harbor dozens of similar polymorphisms that affect processing of a wide range of miRNA precursors. Our results suggest that natural variation in miRNA biogenesis resulting from cis mutations is a common contributor to phenotypic variation in plants.

Nonsense-mediated mRNA decay modulates FLM-dependent thermosensory flowering response in Arabidopsis.

Increasing global temperatures have an impact on flowering, and the underlying mechanisms are just beginning to be unravelled1,2. Elevated temperatures can induce flowering, and different mechanisms that involve either activation or de-repression of FLOWERING LOCUS T (FT) by transcription factor PHYTOCHROME INTERACTING FACTOR 4 (PIF4) or the FLOWERING LOCUS M (FLM)–SHORT VEGETATIVE PHASE (SVP) complex, respectively, have been suggested to be involved3–6. Thermosensitivity in flowering has been mapped to FLM5, which encodes a floral repressor7,8. FLM undergoes alternative splicing8 and it has been suggested that temperature-dependent alternative splicing leads to differential accumulation of the FLM-β and FLM-δ transcripts, encoding proteins with antagonistic effects, and that their ratio determines floral transition4. Here we show that high temperatures downregulate FLM expression by alternative splicing coupled with nonsense-mediated mRNA decay (AS-NMD). We identify thermosensitive splice sites in FLM and show that the primary effect of temperature is explained by an increase in NMD target transcripts. We also show that flm is epistatic to pif4, which suggests that most of the PIF4 effects are FLM dependent. Our findings suggest a model in which the loss of the floral repressor FLM occurs through mRNA degradation in response to elevated temperatures, signifying a role for AS-NMD in conferring environmental responses in plants.