Ozone and nitrogen dioxide regulate similar gene expression responses in Arabidopsis but natural variation in the extent of cell death is likely controlled by different genetic loci.

High doses of ozone (O3) and nitrogen dioxide (NO2) cause damage and cell death in plants. These two gases are among the most harmful air pollutants for ecosystems and therefore it is important to understand how plant resistance or sensitivity to these gases work at the molecular level and its genetic control. We compared transcriptome data from O3 and NO2 fumigations to other cell death related treatments, as well as individual marker gene transcript level in different Arabidopsis thaliana accessions. Our analysis revealed that O3 and NO2 trigger very similar gene expression responses that include genes involved in pathogen resistance, cell death and ethylene signaling. However, we also identified exceptions, for example RBOHF encoding a reactive oxygen species producing RESPIRATORY BURST OXIDASE PROTEIN F. This gene had increased transcript levels by O3 but decreased transcript levels by NO2, showing that plants can identify each of the gases separately and activate distinct signaling pathways. To understand the genetics, we conducted a genome wide association study (GWAS) on O3 and NO2 tolerance of natural Arabidopsis accessions. Sensitivity to both gases seem to be controlled by several independent small effect loci and we did not find an overlap in the significantly associated regions. Further characterization of the GWAS candidate loci identified new regulators of O3 and NO2 induced cell death including ABH1, a protein that functions in abscisic acid signaling, mRNA splicing and miRNA processing. The GWAS results will facilitate further characterization of the control of programmed cell death and differences between oxidative and nitrosative stress in plants.

Ozone responses in Arabidopsis: beyond stomatal conductance.

Tropospheric ozone (O3) is a major air pollutant that decreases yield of important crops worldwide. Despite long-lasting research of its negative effects on plants, there are many gaps in our knowledge on how plants respond to O3. In this study, we used natural variation in the model plant Arabidopsis (Arabidopsis thaliana) to characterize molecular and physiological mechanisms underlying O3 sensitivity. A key parameter in models for O3 damage is stomatal uptake. Here we show that the extent of O3 damage in the sensitive Arabidopsis accession Shahdara (Sha) does not correspond with O3 uptake, pointing toward stomata-independent mechanisms for the development of O3 damage. We compared tolerant (Col-0) versus sensitive accessions (Sha, Cvi-0) in assays related to photosynthesis, cell death, antioxidants, and transcriptional regulation. Acute O3 exposure increased cell death, development of lesions in the leaves, and decreased photosynthesis in sensitive accessions. In both Sha and Cvi-0, O3-induced lesions were associated with decreased maximal chlorophyll fluorescence and low quantum yield of electron transfer from Photosystem II to plastoquinone. However, O3-induced repression of photosynthesis in these two O3-sensitive accessions developed in different ways. We demonstrate that O3 sensitivity in Arabidopsis is influenced by genetic diversity given that Sha and Cvi-0 developed accession-specific transcriptional responses to O3. Our findings advance the understanding of plant responses to O3 and set a framework for future studies to characterize molecular and physiological mechanisms allowing plants to respond to high O3 levels in the atmosphere as a result of high air pollution and climate change.

Interaction of methyl viologen-induced chloroplast and mitochondrial signalling in Arabidopsis.

Reactive oxygen species (ROS) are key signalling intermediates in plant metabolism, defence, and stress adaptation. In plants, both the chloroplast and mitochondria are centres of metabolic control and ROS production, which coordinate stress responses in other cell compartments. The herbicide and experimental tool, methyl viologen (MV) induces ROS generation in the chloroplast under illumination, but is also toxic in non-photosynthetic organisms. We used MV to probe plant ROS signalling in compartments other than the chloroplast. Taking a genetic approach in the model plant Arabidopsis (Arabidopsis thaliana), we used natural variation, QTL mapping, and mutant studies with MV in the light, but also under dark conditions, when the chloroplast electron transport is inactive. These studies revealed a light-independent MV-induced ROS-signalling pathway, suggesting mitochondrial involvement. Mitochondrial Mn SUPEROXIDE DISMUTASE was required for ROS-tolerance and the effect of MV was enhanced by exogenous sugar, providing further evidence for the role of mitochondria. Mutant and hormone feeding assays revealed roles for stress hormones in organellar ROS-responses. The radical-induced cell death1 mutant, which is tolerant to MV-induced ROS and exhibits altered mitochondrial signalling, was used to probe interactions between organelles. Our studies suggest that mitochondria are involved in the response to ROS induced by MV in plants.

High-throughput sequencing data and the impact of plant gene annotation quality.

The use of draft genomes of different species and re-sequencing of accessions and populations are now common tools for plant biology research. The de novo assembled draft genomes make it possible to identify pivotal divergence points in the plant lineage and provide an opportunity to investigate the genomic basis and timing of biological innovations by inferring orthologs between species. Furthermore, re-sequencing facilitates the mapping and subsequent molecular characterization of causative loci for traits, such as those for plant stress tolerance and development. In both cases high-quality gene annotation-the identification of protein-coding regions, gene promoters, and 5'- and 3'-untranslated regions-is critical for investigation of gene function. Annotations are constantly improving but automated gene annotations still require manual curation and experimental validation. This is particularly important for genes with large introns, genes located in regions rich with transposable elements or repeats, large gene families, and segmentally duplicated genes. In this opinion paper, we highlight the impact of annotation quality on evolutionary analyses, genome-wide association studies, and the identification of orthologous genes in plants. Furthermore, we predict that incorporating accurate information from manual curation into databases will dramatically improve the performance of automated gene predictors.

Genome sequencing and population genomic analyses provide insights into the adaptive landscape of silver birch.

Silver birch (Betula pendula) is a pioneer boreal tree that can be induced to flower within 1 year. Its rapid life cycle, small (440-Mb) genome, and advanced germplasm resources make birch an attractive model for forest biotechnology. We assembled and chromosomally anchored the nuclear genome of an inbred B. pendula individual. Gene duplicates from the paleohexaploid event were enriched for transcriptional regulation, whereas tandem duplicates were overrepresented by environmental responses. Population resequencing of 80 individuals showed effective population size crashes at major points of climatic upheaval. Selective sweeps were enriched among polyploid duplicates encoding key developmental and physiological triggering functions, suggesting that local adaptation has tuned the timing of and cross-talk between fundamental plant processes. Variation around the tightly-linked light response genes PHYC and FRS10 correlated with latitude and longitude and temperature, and with precipitation for PHYC. Similar associations characterized the growth-promoting cytokinin response regulator ARR1, and the wood development genes KAK and MED5A.

Transcriptomics and functional genomics of ROS-induced cell death regulation by RADICAL-INDUCED CELL DEATH1.

Plant responses to changes in environmental conditions are mediated by a network of signaling events leading to downstream responses, including changes in gene expression and activation of cell death programs. Arabidopsis thaliana RADICAL-INDUCED CELL DEATH1 (RCD1) has been proposed to regulate plant stress responses by protein-protein interactions with transcription factors. Furthermore, the rcd1 mutant has defective control of cell death in response to apoplastic reactive oxygen species (ROS). Combining transcriptomic and functional genomics approaches we first used microarray analysis in a time series to study changes in gene expression after apoplastic ROS treatment in rcd1. To identify a core set of cell death regulated genes, RCD1-regulated genes were clustered together with other array experiments from plants undergoing cell death or treated with various pathogens, plant hormones or other chemicals. Subsequently, selected rcd1 double mutants were constructed to further define the genetic requirements for the execution of apoplastic ROS induced cell death. Through the genetic analysis we identified WRKY70 and SGT1b as cell death regulators functioning downstream of RCD1 and show that quantitative rather than qualitative differences in gene expression related to cell death appeared to better explain the outcome. Allocation of plant energy to defenses diverts resources from growth. Recently, a plant response termed stress-induced morphogenic response (SIMR) was proposed to regulate the balance between defense and growth. Using a rcd1 double mutant collection we show that SIMR is mostly independent of the classical plant defense signaling pathways and that the redox balance is involved in development of SIMR.

Complex genetic effects on early vegetative development shape resource allocation differences between Arabidopsis lyrata populations.

Costs of reproduction due to resource allocation trade-offs have long been recognized as key forces in life history evolution, but little is known about their functional or genetic basis. Arabidopsis lyrata, a perennial relative of the annual model plant A. thaliana with a wide climatic distribution, has populations that are strongly diverged in resource allocation. In this study, we evaluated the genetic and functional basis for variation in resource allocation in a reciprocal transplant experiment, using four A. lyrata populations and F2 progeny from a cross between North Carolina (NC) and Norway parents, which had the most divergent resource allocation patterns. Local alleles at quantitative trait loci (QTL) at a North Carolina field site increased reproductive output while reducing vegetative growth. These QTL had little overlap with flowering date QTL. Structural equation models incorporating QTL genotypes and traits indicated that resource allocation differences result primarily from QTL effects on early vegetative growth patterns, with cascading effects on later vegetative and reproductive development. At a Norway field site, North Carolina alleles at some of the same QTL regions reduced survival and reproductive output components, but these effects were not associated with resource allocation trade-offs in the Norway environment. Our results indicate that resource allocation in perennial plants may involve important adaptive mechanisms largely independent of flowering time. Moreover, the contributions of resource allocation QTL to local adaptation appear to result from their effects on developmental timing and its interaction with environmental constraints, and not from simple models of reproductive costs.

Investigating incipient speciation in Arabidopsis lyrata from patterns of transmission ratio distortion.

Our understanding of the development of intrinsic reproductive isolation is still largely based on theoretical models and thorough empirical studies on a small number of species. Theory suggests that reproductive isolation develops through accumulation of epistatic genic incompatibilities, also known as Bateson-Dobzhansky-Muller (BDM) incompatibilities. We can detect these from marker transmission ratio distortion (TRD) in hybrid progenies of crosses between species or populations, where TRD is expected to result from selection against heterospecific allele combinations in hybrids. TRD may also manifest itself because of intragenomic conflicts or competition between gametes or zygotes. We studied early stage speciation in Arabidopsis lyrata by investigating patterns of TRD across the genome in F2 progenies of three reciprocal crosses between four natural populations. We found that the degree of TRD increases with genetic distance between crossed populations, but also that reciprocal progenies may differ substantially in their degree of TRD. Chromosomes AL6 and especially AL1 appear to be involved in many single- and two-locus distortions, but the location and source of TRD vary between crosses and between reciprocal progenies. We also found that the majority of single- and two-locus TRD appears to have a gametic, as opposed to zygotic, origin. Thus, while theory on BDM incompatibilities is typically illustrated with derived nuclear alleles proving incompatible in hybrid zygotes, our results suggest a prominent role for distortions emerging before zygote formation.

Genetic changes in flowering and morphology in response to adaptation to a high-latitude environment in Arabidopsis lyrata.

BACKGROUND AND AIMS: The adaptive plastic reactions of plant populations to changing climatic factors, such as winter temperatures and photoperiod, have changed during range shifts after the last glaciation. Timing of flowering is an adaptive trait regulated by environmental cues. Its genetics has been intensively studied in annual plants, but in perennials it is currently not well characterized. This study examined the genetic basis of differentiation in flowering time, morphology, and their plastic responses to vernalization in two locally adapted populations of the perennial Arabidopsis lyrata: (1) to determine whether the two populations differ in their vernalization responses for flowering phenology and morphology; and (2) to determine the genomic areas governing differentiation and vernalization responses. METHODS: Two A. lyrata populations, from central Europe and Scandinavia, were grown in growth-chamber conditions with and without cold treatment. A QTL analysis was performed to find genomic regions that interact with vernalization. KEY RESULTS: The population from central Europe flowered more rapidly and invested more in inflorescence growth than the population from alpine Scandinavia, especially after vernalization. The alpine population had consistently a low number of inflorescences and few flowers, suggesting strong constraints due to a short growing season, but instead had longer leaves and higher leaf rosettes. QTL mapping in the F2 population revealed genomic regions governing differentiation in flowering time and morphology and, in some cases, the allelic effects from the two populations on a trait were influenced by vernalization (QTL × vernalization interactions). CONCLUSIONS: The results indicate that many potentially adaptive genetic changes have occurred during colonization; the two populations have diverged in their plastic responses to vernalization in traits closely connected to fitness through changes in many genomic areas.

Genetic basis of local adaptation and flowering time variation in Arabidopsis lyrata.

Understanding how genetic variation at individual loci contributes to adaptation of populations to different local environments is an important topic in modern evolutionary biology. To date, most evidence has pointed to conditionally neutral quantitative trait loci (QTL) showing fitness effects only in some environments, while there has been less evidence for single-locus fitness trade-offs. At QTL underlying local adaptation, alleles from the local population are expected to show a fitness advantage. Cytoplasmic genomes also can have a role in local adaptation, but the role of cytonuclear interactions in adaptive differentiation has remained largely unknown. We mapped genomic regions underlying adaptive differentiation in multiple fitness components and flowering time in diverged populations of a perennial plant Arabidopsis lyrata. Experimental hybrids for this purpose were grown in natural field conditions of the parental populations in Norway and North Carolina (NC), USA, and in the greenhouse. We found QTL where high fitness and early flowering were associated with local alleles, indicating a role of different selection pressures in phenotypic differentiation. At two QTL regions, a fitness component showing local adaptation between the parental populations also showed signs of putative fitness trade-offs. Beneficial dominance effects of conditionally neutral QTL for different fitness components resulted in hybrid vigour at the Norwegian site in the F(2) hybrids. We also found that cytoplasmic genomes contributed to local adaptation and hybrid vigour by interacting with nuclear QTL, but these interactions did not show evidence for cytonuclear coadaptation (high fitness of local alleles combined with the local cytoplasm).

Nuclear-cytoplasmic interactions reduce male fertility in hybrids of Arabidopsis lyrata subspecies.

We examined the level of postzygotic reproductive isolation in F(1) and F(2) hybrids of reciprocal crosses between the Arabidopsis lyrata subspecies lyrata (North American) and petraea (European). Our main results are: first, the percentage of fertile pollen was significantly reduced in the F(1) and F(2) compared to the parental populations. Second, mean pollen fertility differed markedly between reciprocal crosses: 84% in the F(2) with ssp. lyrata cytoplasm and 61% in the F(2) with ssp. petraea cytoplasm. Third, 17% of the F(2) with ssp. petraea cytoplasm showed male sterility (produced less than 30 pollen grains in our subsample). The hybrids were female fertile. We used QTL mapping to find the genomic regions that determine pollen fertility and that restore cytoplasmic male sterility (CMS). In the F(2) with ssp. lyrata cytoplasm, an epistatic pair of QTLs was detected. In the reciprocal F(2) progeny, four QTLs demonstrated within-population polymorphism for hybrid male sterility. In addition, in the F(2) with ssp. petraea cytoplasm, there was a strong male fertility restorer locus on chromosome 2 where a cluster of CMS restorer gene-related PPR genes have been found in A. lyrata. Our results underline the importance of cytonuclear interactions in understanding genetics of the early stages of speciation.

Construction and evaluation of a self-contained index for assessment of diet quality.

AIMS: To construct and validate a self-contained index for the evaluation of a health-promoting diet in adults. METHODS: Participants (n = 103) were healthy volunteer adults aged 20 to 64 years. A food consumption questionnaire containing 55 questions was formulated and evaluated against seven-day food records. Key questions best reflecting the health-promoting diet, defined in nutrition recommendations, were identified by correlation and ROC analyses in comparison to calculated food and nutrient intakes from the food records. A shorter questionnaire was scored to compile an Index of Diet Quality (IDQ). RESULTS: Based on ROC analyses 18 questions were sufficient to describe the health-promoting diet and comprised the index. IDQ had a sensitivity of 67% and a specificity of 71%. The IDQ score reflected dietary intake, shown as statistically significant correlations between higher IDQ scores and higher intakes of protein (r = 0.35), fibre (r = 0.42), calcium (r = 0.39), iron (r = 0.31), vitamin C (r = 0.31) and a higher ratio of unsaturated to saturated fatty acids (r = 0.23) and a lower intake of saturated fatty acids (r = -0.22) and saccharose (r = -0.25). CONCLUSIONS: IDQ reflects dietary intake of key foods and nutrients associated with health and depicts adherence to dietary recommendations. It is applicable in nutritional studies where diet in its entirety is of interest and also in large-scale studies, being fast in execution with analysis free of complex calculations.

Comparing the linkage maps of the close relatives Arabidopsis lyrata and A. thaliana.

We have constructed a genetic map of Arabidopsis lyrata, a self-incompatible relative of the plant model species A. thaliana. A. lyrata is a diploid (n = 8) species that diverged from A. thaliana (n = 5) approximately 5 MYA. Mapping was conducted in a full-sib progeny of two unrelated F(1) hybrids between two European populations of A. lyrata ssp. petraea. We used the least-squares method of the Joinmap program for map construction. The gross chromosomal differences between the two species were most parsimoniously explained with three fusions, two reciprocal translocations, and one inversion. The total map length was 515 cM, and the distances were 12% larger than those between corresponding markers in the linkage map of A. thaliana. The 72 markers, consisting of microsatellites and gene-based markers, were spaced on average every 8 cM. Transmission ratio distortion was extensive, and most distortions were specific to each reciprocal cross, suggesting cytoplasmic interactions. We estimate locations and most probable genotype frequencies of transmission ratio distorting loci (TRDL) with a Bayesian method and discuss the possible reasons for the observed distortions.