Arabidopsis species deploy distinct strategies to cope with drought stress.

BACKGROUND AND AIMS: Water limitation is an important determinant of the distribution, abundance and diversity of plant species. Yet, little is known about how the response to limiting water supply changes among closely related plant species with distinct ecological preferences. Comparison of the model annual species Arabidopsis thaliana with its close perennial relatives A. lyrata and A. halleri, can help disentangle the molecular and physiological changes contributing to tolerance and avoidance mechanisms, because these species must maintain tolerance and avoidance mechanisms to increase long-term survival, but they are exposed to different levels of water stress and competition in their natural habitat. METHODS: A dry-down experiment was conducted to mimic a period of missing precipitation. The covariation of a progressive decrease in soil water content (SWC) with various physiological and morphological plant traits across a set of representative genotypes in A. thaliana, A. lyrata and A. halleri was quantified. Transcriptome changes to soil dry-down were further monitored. KEY RESULTS: The analysis of trait covariation demonstrates that the three species differ in the strategies they deploy to respond to drought stress. Arabidopsis thaliana showed a drought avoidance reaction but failed to survive wilting. Arabidopsis lyrata efficiently combined avoidance and tolerance mechanisms. In contrast, A. halleri showed some degree of tolerance to wilting but it did not seem to protect itself from the stress imposed by drought. Transcriptome data collected just before plant wilting and after recovery corroborated the phenotypic analysis, with A. lyrata and A. halleri showing a stronger activation of recovery- and stress-related genes, respectively. CONCLUSIONS: The response of the three Arabidopsis species to soil dry-down reveals that they have evolved distinct strategies to face drought stress. These strategic differences are in agreement with the distinct ecological priorities of the stress-tolerant A. lyrata, the competitive A. halleri and the ruderal A. thaliana.

Phenotypic and metabolic responses to drought and salinity of four contrasting lentil accessions.

Drought and salinity are among the major abiotic stresses which, often inter-relatedly, adversely affect plant growth and productivity. Plant stress responses depend on the type of stress, on its intensity, on the species, and also on the genotype. Different accessions of a species may have evolved different mechanisms to cope with stress and to complete their life cycles. This study is focused on lentil, an important Mediterranean legume with high quality protein for the human diet. The effects of salinity and drought on germination and early growth of Castelluccio di Norcia (CAST), Pantelleria (PAN), Ustica (UST), and Eston (EST) accessions were evaluated to identify metabolic and phenotypic traits related to drought and/or salinity stress tolerance. The results showed a relationship between imposed stresses and performance of the cultivars. According to germination frequencies, the accession ranking was as follows: NaCl resistant > susceptible, PAN > UST > CAST > EST; polyethylene glycol (PEG) resistant > susceptible, CAST > UST > EST > PAN. Seedling tolerance rankings were: NaCl resistant > susceptible, CAST ≈ UST > PAN ≈ EST; PEG resistant > susceptible, CAST > EST ≈ UST > PAN. Changes in the metabolite profiles, mainly quantitative rather than qualitative, were observed in the same cultivar in respect to the treatments, and among the cultivars under the same treatment. Metabolic differences in the stress tolerance of the different genotypes were related to a reduction in the levels of tricarboxylic acid (TCA) cycle intermediates. The relevant differences, between the most NaCl-tolerant genotype (PAN) and the most sensitive one (EST) were related to the decrease in the threonic acid level. Stress-specific metabolite indicators were also identified: ornithine and asparagine as markers of drought stress and alanine and homoserine as markers of salinity stress.

Dent and Flint maize diversity panels reveal important genetic potential for increasing biomass production.

KEY MESSAGE: Genetic and phenotypic analysis of two complementary maize panels revealed an important variation for biomass yield. Flowering and biomass QTL were discovered by association mapping in both panels. The high whole plant biomass productivity of maize makes it a potential source of energy in animal feeding and biofuel production. The variability and the genetic determinism of traits related to biomass are poorly known. We analyzed two highly diverse panels of Dent and Flint lines representing complementary heterotic groups for Northern Europe. They were genotyped with the 50 k SNP-array and phenotyped as hybrids (crossed to a tester of the complementary pool) in a western European field trial network for traits related to flowering time, plant height, and biomass. The molecular information revealed to be a powerful tool for discovering different levels of structure and relatedness in both panels. This study revealed important variation and potential genetic progress for biomass production, even at constant precocity. Association mapping was run by combining genotypes and phenotypes in a mixed model with a random polygenic effect. This permitted the detection of significant associations, confirming height and flowering time quantitative trait loci (QTL) found in literature. Biomass yield QTL were detected in both panels but were unstable across the environments. Alternative kinship estimator only based on markers unlinked to the tested SNP increased the number of significant associations by around 40% with a satisfying control of the false positive rate. This study gave insights into the variability and the genetic architectures of biomass-related traits in Flint and Dent lines and suggests important potential of these two pools for breeding high biomass yielding hybrid varieties.

A complete BAC-based physical map of the Arabidopsis thaliana genome.

Arabidopsis thaliana is a small flowering plant that serves as the major model system in plant molecular genetics. The efforts of many scientists have produced genetic maps that provide extensive coverage of the genome (http://genome-www. stanford.edu/Arabidopsis/maps.html). Recently, detailed YAC, BAC, P1 and cosmid-based physical maps (that is, representations of genomic regions as sets of overlapping clones of corresponding libraries) have been established that extend over wide genomic areas ranging from several hundreds of kilobases to entire chromosomes. These maps provide an entry to gain deeper insight into the A. thaliana genome structure. A. thaliana has been chosen as the subject of the first large-scale project intended to determine the full genome sequence of a plant. This sequencing project, together with the increasing interest in map-based gene cloning, has highlighted the requirement for a complete and accurate physical map of this plant species. To supply the scientific community with a high-quality resource, we present here a complete physical map of A. thaliana using essentially the IGF BAC library. The map consists of 27 contigs that cover the entire genome, except for the presumptive centromeric regions, nucleolar organization regions (NOR) and telomeric areas. This is the first reported map of a complex organism based entirely on BAC clones and it represents the most homogeneous and complete physical map established to date for any plant genome. Furthermore, the analysis performed here serves as a model for an efficient physical mapping procedure using BAC clones that can be applied to other complex genomes.

Symptomatic relief from at-home use of activated Bifidobacterium infantis EVC001 probiotic in infants: results from a consumer survey on the effects on diaper rash, colic symptoms, and sleep.

The gut microbiome during infancy is directly involved in the digestion of human milk, development of the immune system, and long-term health outcomes. Gut dysbiosis in early life has been linked to multiple short-term ailments, from diaper dermatitis and poor stooling habits, to poor sleep and fussiness, with mixed results in the scientific literature on the efficacy of probiotics for symptom resolution. Despite the growing interest in probiotics for consumer use, observed symptomatic relief is rarely documented. This study aims to evaluate observed symptomatic relief from at-home use of activated Bifidobacterium infantis EVC001 in infants. Consumer feedback was collected over a 2-year period via a 30-day post-purchase online survey of B. infantis EVC001 (Evivo®) customers. Outcome measures included observed changes in diaper rash, symptoms of colic, and sleep behaviours in infants fed B. infantis EVC001. A total of 1,621 respondents completed the survey. Before purchasing B. infantis EVC001, the majority of respondents visited the product website, researched infant probiotics online, or consulted with their doctor or other healthcare professional. Of the participants whose infants had ever experienced diaper rash, 72% (n=448) reported improvements, and 57% of those reported complete resolution of this problem. Of those who responded to questions about gassiness/fussiness, naptime sleep, and night-time sleep behaviours, 63% (n=984), 33% (n=520), and 52% (n=806) reported resolution or improvements, respectively. Although clinical data regarding probiotic use are often inconclusive for symptom resolution, home use of B. infantis EVC001 in infants improved diaper rash, gassiness/fussiness, and sleep quality within the first week of use in a significant number of respondents who engaged in a voluntary post-purchase survey. These outcomes may be a result of the unique genetic capacity of B. infantis EVC001 to colonise the infant gut highlighting the importance of strain selection in evaluating the effects of probiotic products.

A tale of dwarfs and drugs: brassinosteroids to the rescue.

Brassinosteroids (BRs), a specific class of low abundance plant steroids, are capable of eliciting strong growth responses and a variety of physiological changes through exogenous application to plants. Recently, BRs gained general acceptance as important regulators (hormones) of plant growth and development through genetic and molecular identification and characterization of genes involved in BR biosynthesis or response in Arabidopsis, pea and tomato. This major advance in the molecular genetics of BR biosynthesis and mode of action disclosed another case of amazing functional conservation of signalling molecules utilized in plants and animals, and provided a set of extremely valuable tools for the functional analysis of BRs.

Metabolite profiling for plant functional genomics.

Multiparallel analyses of mRNA and proteins are central to today's functional genomics initiatives. We describe here the use of metabolite profiling as a new tool for a comparative display of gene function. It has the potential not only to provide deeper insight into complex regulatory processes but also to determine phenotype directly. Using gas chromatography/mass spectrometry (GC/MS), we automatically quantified 326 distinct compounds from Arabidopsis thaliana leaf extracts. It was possible to assign a chemical structure to approximately half of these compounds. Comparison of four Arabidopsis genotypes (two homozygous ecotypes and a mutant of each ecotype) showed that each genotype possesses a distinct metabolic profile. Data mining tools such as principal component analysis enabled the assignment of "metabolic phenotypes" using these large data sets. The metabolic phenotypes of the two ecotypes were more divergent than were the metabolic phenotypes of the single-loci mutant and their parental ecotypes. These results demonstrate the use of metabolite profiling as a tool to significantly extend and enhance the power of existing functional genomics approaches.

Recent advances in brassinosteroid molecular genetics.

The importance of brassinosteroids (BRs, a specific class of ecdysone-like plant steroids) as essential endogenous regulators of growth and development is demonstrated through a growing number of well characterised Arabidopsis, pea, and tomato mutants deficient in BR biosynthesis or BR response. Thus, a rapid advancement in understanding the molecular genetics of BR biosynthesis and mode of action can be witnessed, which will be further enhanced through the availability of a set of extremely valuable molecular tools for the analysis of the biological function of BRs.

Stomatal cell biology.

In the past two years, major advances in our knowledge of the cellular events involved in the formation of stomatal complexes, particularly those responsible for the establishment of the stomatal pattern, have been made. These events are altered in the Arabidopsis mutants sdd1-1, tmm and flp. Molecular cloning of the SDD1 gene initiated the elucidation of the underlying molecular mechanisms. The proposed function of SDD1 as a processing protease provides a hint towards the involvement of a proteinaceous signal in the formation of stomatal complexes.

Molecular analysis of brassinosteroid action.

Brassinosteroids (BRs) are steroidal plant hormones with important regulatory roles in various physiological processes, including growth, xylem differentiation, disease resistance, and stress tolerance. Several components of the BR signal transduction pathway have been identified. The extracellular domains of receptor kinases such as BRI1 perceive BRs and transduce the signal via intracellular kinase domains. Within the cell further kinases and phosphatases determine the phosphorylation status of transcription factors such as BES1 and BZR1. These factors mediate major BR effects. Studies of BR-regulated genes shed light on the molecular mode of BR action. Genes encoding cell-wall-modifying enzymes, enzymes of the BR biosynthetic pathway, transcription factors, and proteins involved in primary and secondary metabolism are subject to BR-regulation. Gene expression data also point at interactions with other phytohormones and a role of BR in stress responses. This article gives a survey of the BR-signaling pathway. Two BR-responsive genes, OPR3 and EXO, are described in detail.

Blockade of the PD-1/PD-L1 axis augments lysis of AML cells by the CD33/CD3 BiTE antibody construct AMG 330: reversing a T-cell-induced immune escape mechanism.

Bispecific T-cell engagers (BiTEs) are very effective in recruiting and activating T cells. We tested the cytotoxicity of the CD33/CD3 BiTE antibody construct AMG 330 on primary acute myeloid leukemia (AML) cells ex vivo and characterized parameters contributing to antileukemic cytolytic activity. The E:T ratio and the CD33 expression level significantly influenced lysis kinetics in long-term cultures of primary AML cells (n=38). AMG 330 induced T-cell-mediated proinflammatory conditions, favoring the upregulation of immune checkpoints on target and effector cells. Although not constitutively expressed at the time of primary diagnosis (n=123), PD-L1 was strongly upregulated on primary AML cells upon AMG 330 addition to ex vivo cultures (n=27, P<0.0001). This phenomenon was cytokine-driven as the sole addition of interferon (IFN)-γ and tumor necrosis factor-α also induced expression. Through blockade of the PD-1/PD-L1 interaction, AMG 330-mediated lysis (n=9, P=0.03), T-cell proliferation (n=9, P=0.01) and IFN-γ secretion (n=8, P=0.008) were significantly enhanced. The combinatorial approach was most beneficial in settings of protracted AML cell lysis. Taken together, we have characterized a critical resistance mechanism employed by primary AML cells under AMG 330-mediated proinflammatory conditions. Our results support the evaluation of checkpoint molecules in upcoming clinical trials with AMG 330 to enhance BiTE antibody construct-mediated cytotoxicity.

Integrated studies on plant biology using multiparallel techniques.

Plant biology, especially the fields of molecular genetics and molecular physiology, is currently undergoing a change in paradigm from 'vertical' analysis of the role(s) of one or a few genes to 'horizontal' holistic approaches, studying the function of many or even all of the genes of an organism simultaneously. This change is leading us beyond genomes to transcriptomes, proteomes and metabalomes, and to an understanding of life at an entirely new level. Profiling strategies are putting this change into effect through the generation of large amounts of data, requiring that current bioinformatic approaches adapt and grow in order to make the most of these data.

Brassinosteroid signaling in plants.

In animals and humans, steroid hormones (SHs) regulate gene transcription via the binding of nuclear receptors. In addition, rapid nongenomic effects of steroids occur and appear to be mediated by plasma-membrane receptors. Plants also use steroids as signaling molecules. These brassinosteroids (BRs) show structural similarity to the SHs of vertebrates and insects. Plant mutants defective in brassinosteroid biosynthesis or perception exhibit dwarfism and reduced fertility, and reveal the need for BRs during growth and development. BR signaling in Arabidopsis thaliana and rice (Oryza sativa) - dicotyledonous and monocotyledonous models, respectively - is mediated by the receptor kinases BRI1 and OsBRI1. The extracellular domain of BRI1 perceives BRs and the signal is mediated via an intracellular kinase domain that autophosphorylates Ser and Thr residues and apparently has the potential to phosphorylate other substrates. BRI1 transduces steroid signals across the plasma membrane and mediates genomic effects.

Hypoxia during early developmental period induces long-term changes in the dopamine content and release in a mesencephalic cell culture.

The present study was conducted to elucidate the long-term effects of exposure to hypoxia of dopaminergic neurons during the early developmental period. Primary mesencephalic cell cultures prepared from fetal rats and containing 0.5-2% of dopaminergic neurons were exposed to hypoxia between in vitro days 1 and 6, the putative critical developmental period. Changes in the content, release and uptake of dopamine were found to depend on the degree of hypoxia and on the duration of exposure. Following moderate hypoxia (7 h, 5% O2) on two consecutive days between in vitro days 1 and 3, the cultures showed a small increase in the dopamine levels, by 16%. After severe hypoxia (0% O2/95% N2 for 24 h), during the same time window, the cellular dopamine content was elevated by 100%. Moreover, severe hypoxia produced long-lasting modulations of the dopaminergic system. On in vitro day 14, cells exhibited increased levels of 3,4-dihydroxyphenylacetic acid and homovanillic acid (by 34% and 55%, respectively), and elevations of both the spontaneous and potassium-stimulated dopamine release by 70%. The dopamine transport and metabolism of cells exposed to hypoxia between in vitro days 4 and 6 remained unchanged with regard to long-term effects. The present study provides strong evidence for the induction of long-term changes in dopaminergic cells due to hypoxia during the critical developmental period in mesencephalic culture. The developmental period capable of inducing long-lasting changes in dopamine metabolism is restricted to in vitro days 1-3.

Brassinosteroids from seeds of Arabidopsis thaliana.

Extracts of seeds of Arabidopsis thaliana (ecotype 24) were analysed for the presence of free and conjugated brassinosteroids. 24-epi-Brassinolide (ca 220 ng kg-1) and castasterone (ca 360 ng kg-1) could be isolated and unambiguously identified as native brassinosteroids by GC/MS.

Maximizing the reliability of genomic selection by optimizing the calibration set of reference individuals: comparison of methods in two diverse groups of maize inbreds (Zea mays L.).

Genomic selection refers to the use of genotypic information for predicting breeding values of selection candidates. A prediction formula is calibrated with the genotypes and phenotypes of reference individuals constituting the calibration set. The size and the composition of this set are essential parameters affecting the prediction reliabilities. The objective of this study was to maximize reliabilities by optimizing the calibration set. Different criteria based on the diversity or on the prediction error variance (PEV) derived from the realized additive relationship matrix-best linear unbiased predictions model (RA-BLUP) were used to select the reference individuals. For the latter, we considered the mean of the PEV of the contrasts between each selection candidate and the mean of the population (PEVmean) and the mean of the expected reliabilities of the same contrasts (CDmean). These criteria were tested with phenotypic data collected on two diversity panels of maize (Zea mays L.) genotyped with a 50k SNPs array. In the two panels, samples chosen based on CDmean gave higher reliabilities than random samples for various calibration set sizes. CDmean also appeared superior to PEVmean, which can be explained by the fact that it takes into account the reduction of variance due to the relatedness between individuals. Selected samples were close to optimality for a wide range of trait heritabilities, which suggests that the strategy presented here can efficiently sample subsets in panels of inbred lines. A script to optimize reference samples based on CDmean is available on request.