Quantitative trait loci analysis of hormone levels in Arabidopsis roots.

Quantitative trait loci (QTL) analyses for five groups of hormones, including cytokinins in Arabidopsis roots were performed using recombinant inbred lines (Ler×Cvi). Significant QTLs were detected for cytokinins, jasmonic acid and salicylic acid. Separate analysis of two sub-populations, viz., vegetative and flowering plants revealed that many of the QTLs were development-specific. Using near-isogenic lines, several significant QTLs were confirmed; three co-localized QTL regions were responsible for determining several cytokinin metabolites. Using a knock-out plant, a functional role of zeatin N-glucosyltransferase gene (UGT76C2) underlying a large-effect QTL for levels of tZ-N-glucosides and tZRMP was evaluated in the metabolism of cytokinins. Pleotropic effects of this gene were found for cytokinin levels in both roots and leaves, but significant changes of morphological traits were observed only in roots. Hormone QTL analysis reveals development-specific and organ-dependent aspects of the regulation of plant hormone content and metabolism.

Phytochrome A Protects Tomato Plants From Injuries Induced by Continuous Light.

Plants perceive and transduce information about light quantity, quality, direction and photoperiod via several photoreceptors and use it to adjust their growth and development. A role for photoreceptors has been hypothesized in the injuries that tomato plants develop when exposed to continuous light as the light spectral distribution influences the injury severity. Up to now, however, only indirect clues suggested that phytochromes (PHY), red/far-red photoreceptors, are involved in the continuous-light-induced injuries in tomato. In this study, therefore, we exposed mutant and transgenic tomato plants lacking or over-expressing phytochromes to continuous light, with and without far-red light enrichment. The results show that PHYA over-expression confers complete tolerance to continuous light regardless the light spectrum. Under continuous light with low far-red content, PHYB1 and PHYB2 diminished and enhanced the injury, respectively, yet the effects were small. These results confirm that phytochrome signaling networks are involved in the induction of injury under continuous light. HIGHLIGHTS: - PHYA over-expression confers tolerance to continuous light regardless the light spectrum.- In the absence of far-red light, PHYB1 slightly diminishes the continuous light-induced injury.- Continuous light down-regulates photosynthesis genes in sensitive tomato lines.

Natural variation of hormone levels in Arabidopsis roots and correlations with complex root architecture.

Studies on natural variation are an important tool to unravel the genetic basis of quantitative traits in plants. Despite the significant roles of phytohormones in plant development, including root architecture, hardly any studies have been done to investigate natural variation in endogenous hormone levels in plants. Therefore, in the present study a range of hormones were quantified in root extracts of thirteen Arabidopsis thaliana accessions using a ultra performance liquid chromatography triple quadrupole mass spectrometer. Root system architecture of the set of accessions was quantified, using a new parameter (mature root unit) for complex root systems, and correlated with the phytohormone data. Significant variations in phytohormone levels among the accessions were detected, but were remarkably small, namely less than three-fold difference between extremes. For cytokinins, relatively larger variations were found for ribosides and glucosides, as compared to the free bases. For root phenotyping, length-related traits-lateral root length and total root length-showed larger variations than lateral root number-related ones. For root architecture, antagonistic interactions between hormones, for example, indole-3-acetic acid to trans-zeatin were detected in correlation analysis. These findings provide conclusive evidence for the presence of natural variation in phytohormone levels in Arabidopsis roots, suggesting that quantitative genetic analyses are feasible.

Sucrose and Starch Content Negatively Correlates with PSII Maximum Quantum Efficiency in Tomato (Solanum lycopersicum) Exposed to Abnormal Light/Dark Cycles and Continuous Light.

Light is most important to plants as it fuels photosynthesis and provides clues about the environment. If provided in unnatural long photoperiods, however, it can be harmful and even lethal. Tomato (Solanum lycopersicum), for example, develops mottled chlorosis and necrosis when exposed to continuous light. Understanding the mechanism of these injuries is valuable, as important pathways regulating photosynthesis, such as circadian, retrograde and light signaling pathways are probably involved. Here, we use non-targeted metabolomics and transcriptomics analysis as well as hypothesis-driven experiments with continuous light-tolerant and -sensitive tomato lines to explore the long-standing proposed role of carbohydrate accumulation in this disorder. Analysis of metabolomics and transcriptomics data reveals a clear effect of continuous light on sugar metabolism and photosynthesis. A strong negative correlation between sucrose and starch content with the severity of continuous light-induced damage quantified as the maximum quantum efficiency of PSII (Fv/Fm) was found across several abnormal light/dark cycles, supporting the hypothesis that carbohydrates play an important role in the continuous light-induced injury. We postulate that the continuous light-induced injury in tomato is caused by down-regulation of photosynthesis, showing characteristics of both cytokinin-regulated senescence and light-modulated retrograde signaling. Molecular mechanisms linking carbohydrate accumulation with down-regulation of carbon-fixing enzymes are discussed.

On the induction of injury in tomato under continuous light: circadian asynchrony as the main triggering factor.

Unlike other species, when tomato plants (Solanum lycopersicum L.) are deprived of at least 8h of darkness per day, they develop a potentially lethal injury. In an effort to understand why continuous light (CL) is injurious to tomato, we tested five factors, which potentially could be responsible for triggering the injury in CL-grown tomato: (i) differences in the light spectral distribution between sunlight and artificial light, (ii) continuous light signalling, (iii) continuous supply of light for photosynthesis, (iv) continuous photo-oxidative pressure and (v) circadian asynchrony - a mismatch between the internal circadian clock frequency and the external light/dark cycles. Our results strongly suggest that continuous-light-induced injury does not result from the unnatural spectral distribution of artificial light nor from the continuity of light per se. Instead, circadian asynchrony seems to be the main factor inducing the CL-induced injury, but the mechanism is not by the earlier hypothesised circadian pattern in sensitivity for photoinhibition. Here, however, we show for the first time diurnal fluctuations in sensitivity to photoinhibition during normal photoperiods. Similarly, we also report for the first time diurnal and circadian rhythms in the maximum quantum efficiency of PSII (Fv/Fm) and the parameter F0.

Genetic architecture of plant stress resistance: multi-trait genome-wide association mapping.

Plants are exposed to combinations of various biotic and abiotic stresses, but stress responses are usually investigated for single stresses only. Here, we investigated the genetic architecture underlying plant responses to 11 single stresses and several of their combinations by phenotyping 350 Arabidopsis thaliana accessions. A set of 214 000 single nucleotide polymorphisms (SNPs) was screened for marker-trait associations in genome-wide association (GWA) analyses using tailored multi-trait mixed models. Stress responses that share phytohormonal signaling pathways also share genetic architecture underlying these responses. After removing the effects of general robustness, for the 30 most significant SNPs, average quantitative trait locus (QTL) effect sizes were larger for dual stresses than for single stresses. Plants appear to deploy broad-spectrum defensive mechanisms influencing multiple traits in response to combined stresses. Association analyses identified QTLs with contrasting and with similar responses to biotic vs abiotic stresses, and below-ground vs above-ground stresses. Our approach allowed for an unprecedented comprehensive genetic analysis of how plants deal with a wide spectrum of stress conditions.

Gene expression and physiological responses associated to stomatal functioning in Rosa×hybrida grown at high relative air humidity.

High relative air humidity (RH≥85%) during growth disturbs stomatal functioning, resulting in excessive water loss in conditions of high evaporative demand. We investigated the expression of nine abscisic acid (ABA)-related genes (involved in ABA biosynthesis, oxidation and conjugation) and two non-ABA related genes (involved in the water stress response) aiming to better understand the mechanisms underlying contrasting stomatal functioning in plants grown at high RH. Four rose genotypes with contrasting sensitivity to high RH (one sensitive, one tolerant and two intermediate) were grown at moderate (62±3%) or high (89±4%) RH. The sensitive genotype grown at high RH showed a significantly higher stomatal conductance (gs) and water loss in response to closing stimuli as compared to the other genotypes. Moreover, high RH reduced the leaf ABA concentration and its metabolites to a greater extent in the sensitive genotype as compared to the tolerant one. The large majority of the studied genes had a relevant role on stomatal functioning (NCED1, UGT75B2, BG2, OST1, ABF3 and Rh-APX) while two others showed a minor contribution (CYP707A3 and BG1) and AAO3, CYP707A1 and DREB1B did not contribute to the tolerance trait. These results show that multiple genes form a highly complex regulatory network acting together towards the genotypic tolerance to high RH.

Genome-Wide Association Mapping and Genomic Prediction Elucidate the Genetic Architecture of Morphological Traits in Arabidopsis.

Quantitative traits in plants are controlled by a large number of genes and their interaction with the environment. To disentangle the genetic architecture of such traits, natural variation within species can be explored by studying genotype-phenotype relationships. Genome-wide association studies that link phenotypes to thousands of single nucleotide polymorphism markers are nowadays common practice for such analyses. In many cases, however, the identified individual loci cannot fully explain the heritability estimates, suggesting missing heritability. We analyzed 349 Arabidopsis accessions and found extensive variation and high heritabilities for different morphological traits. The number of significant genome-wide associations was, however, very low. The application of genomic prediction models that take into account the effects of all individual loci may greatly enhance the elucidation of the genetic architecture of quantitative traits in plants. Here, genomic prediction models revealed different genetic architectures for the morphological traits. Integrating genomic prediction and association mapping enabled the assignment of many plausible candidate genes explaining the observed variation. These genes were analyzed for functional and sequence diversity, and good indications that natural allelic variation in many of these genes contributes to phenotypic variation were obtained. For ACS11, an ethylene biosynthesis gene, haplotype differences explaining variation in the ratio of petiole and leaf length could be identified.

Genome-wide association mapping of time-dependent growth responses to moderate drought stress in Arabidopsis.

Large areas of arable land are often confronted with irregular rainfall resulting in limited water availability for part(s) of the growing seasons, which demands research for drought tolerance of plants. Natural variation was observed for biomass accumulation upon controlled moderate drought stress in 324 natural accessions of Arabidopsis. Improved performance under drought stress was correlated with early flowering and lack of vernalization requirement, indicating overlap in the regulatory networks of flowering time and drought response or correlated responses of these traits to natural selection. In addition, plant size was negatively correlated with relative water content (RWC) independent of the absolute water content (WC), indicating a prominent role for soluble compounds. Growth in control and drought conditions was determined over time and was modelled by an exponential function. Genome-wide association (GWA) mapping of temporal plant size data and of model parameters resulted in the detection of six time-dependent quantitative trait loci (QTLs) strongly associated with drought. Most QTLs would not have been identified if plant size was determined at a single time point. Analysis of earlier reported gene expression changes upon drought enabled us to identify for each QTL the most likely candidates.

GWA Mapping of Anthocyanin Accumulation Reveals Balancing Selection of MYB90 in Arabidopsis thaliana.

Induction of anthocyanin accumulation by osmotic stress was assessed in 360 accessions of Arabidopsis thaliana. A wide range of natural variation, with phenotypes ranging from green to completely red/purple rosettes, was observed. A genome wide association (GWA) mapping approach revealed that sequence diversity in a small 15 kb region on chromosome 1 explained 40% of the variation observed. Sequence and expression analyses of alleles of the candidate gene MYB90 identified a causal polymorphism at amino acid (AA) position 210 of this transcription factor of the anthocyanin biosynthesis pathway. This amino acid discriminates the two most frequent alleles of MYB90. Both alleles are present in a substantial part of the population, suggesting balancing selection between these two alleles. Analysis of the geographical origin of the studied accessions suggests that the macro climate is not the driving force behind positive or negative selection for anthocyanin accumulation. An important role for local climatic conditions is, therefore, suggested. This study emphasizes that GWA mapping is a powerful approach to identify alleles that are under balancing selection pressure in nature.

Genome-Wide Association Mapping of Fertility Reduction upon Heat Stress Reveals Developmental Stage-Specific QTLs in Arabidopsis thaliana.

For crops that are grown for their fruits or seeds, elevated temperatures that occur during flowering and seed or fruit set have a stronger effect on yield than high temperatures during the vegetative stage. Even short-term exposure to heat can have a large impact on yield. In this study, we used Arabidopsis thaliana to study the effect of short-term heat exposure on flower and seed development. The impact of a single hot day (35°C) was determined in more than 250 natural accessions by measuring the lengths of the siliques along the main inflorescence. Two sensitive developmental stages were identified, one before anthesis, during male and female meiosis, and one after anthesis, during fertilization and early embryo development. In addition, we observed a correlation between flowering time and heat tolerance. Genome-wide association mapping revealed four quantitative trait loci (QTLs) strongly associated with the heat response. These QTLs were developmental stage specific, as different QTLs were detected before and after anthesis. For a number of QTLs, T-DNA insertion knockout lines could validate assigned candidate genes. Our findings show that the regulation of complex traits can be highly dependent on the developmental timing.

Genome-wide association mapping of growth dynamics detects time-specific and general quantitative trait loci.

Growth is a complex trait determined by the interplay between many genes, some of which play a role at a specific moment during development whereas others play a more general role. To identify the genetic basis of growth, natural variation in Arabidopsis rosette growth was followed in 324 accessions by a combination of top-view imaging, high-throughput image analysis, modelling of growth dynamics, and end-point fresh weight determination. Genome-wide association (GWA) mapping of the temporal growth data resulted in the detection of time-specific quantitative trait loci (QTLs), whereas mapping of model parameters resulted in another set of QTLs related to the whole growth curve. The positive correlation between projected leaf area (PLA) at different time points during the course of the experiment suggested the existence of general growth factors with a function in multiple developmental stages or with prolonged downstream effects. Many QTLs could not be identified when growth was evaluated only at a single time point. Eleven candidate genes were identified, which were annotated to be involved in the determination of cell number and size, seed germination, embryo development, developmental phase transition, or senescence. For eight of these, a mutant or overexpression phenotype related to growth has been reported, supporting the identification of true positives. In addition, the detection of QTLs without obvious candidate genes implies the annotation of novel functions for underlying genes.

Epigenetic basis of morphological variation and phenotypic plasticity in Arabidopsis thaliana.

Epigenetics is receiving growing attention in the plant science community. Epigenetic modifications are thought to play a particularly important role in fluctuating environments. It is hypothesized that epigenetics contributes to plant phenotypic plasticity because epigenetic modifications, in contrast to DNA sequence variation, are more likely to be reversible. The population of decrease in DNA methylation 1-2 (ddm1-2)-derived epigenetic recombinant inbred lines (epiRILs) in Arabidopsis thaliana is well suited for studying this hypothesis, as DNA methylation differences are maximized and DNA sequence variation is minimized. Here, we report on the extensive heritable epigenetic variation in plant growth and morphology in neutral and saline conditions detected among the epiRILs. Plant performance, in terms of branching and leaf area, was both reduced and enhanced by different quantitative trait loci (QTLs) in the ddm1-2 inherited epigenotypes. The variation in plasticity associated significantly with certain genomic regions in which the ddm1-2 inherited epigenotypes caused an increased sensitivity to environmental changes, probably due to impaired genetic regulation in the epiRILs. Many of the QTLs for morphology and plasticity overlapped, suggesting major pleiotropic effects. These findings indicate that epigenetics contributes substantially to variation in plant growth, morphology, and plasticity, especially under stress conditions.

Expression of auxin synthesis gene tms1 under control of tuber-specific promoter enhances potato tuberization in vitro.

Phytohormones, auxins in particular, play an important role in plant development and productivity. Earlier data showed positive impact of exogenous auxin on potato (Solanum tuberosum L.) tuberization. The aim of this study was to generate potato plants with increased auxin level predominantly in tubers. To this end, a pBinB33-tms1 vector was constructed harboring the Agrobacterium auxin biosynthesis gene tms1 fused to tuber-specific promoter of the class I patatin gene (B33-promoter) of potato. Among numerous independently generated B33:tms1 lines, those without visible differences from control were selected for detailed studies. In the majority of transgenic lines, tms1 gene transcription was detected, mostly in tubers rather than in shoots. Indoleacetic acid (IAA) content in tubers and the auxin tuber-to-shoot ratio were increased in tms1-expressing transformants. The organ-specific increase in auxin synthesis in B33:tms1-transformants accelerated and intensified the process of tuber formation, reduced the dose of carbohydrate supply required for in vitro tuberization, and decreased the photoperiodic dependence of tuber initiation. Overall, a positive correlation was observed between tms1 expression, IAA content in tubers, and stimulation of tuber formation. The revealed properties of B33:tms1 transformants imply an important role for auxin in potato tuberization and offer prospects to magnify potato productivity by a moderate organ-specific enhancement of auxin content.

Continuous-light tolerance in tomato is graft-transferable.

Continuous light induces a potentially lethal injury in domesticated tomato (Solanum lycopersicum) plants. Recently, continuous-light tolerance was reported in several wild tomato species, yet the molecular mechanisms underpinning tolerance/sensitivity are still elusive. Here, we investigated from which part of the plant continuous-light tolerance originates and whether this trait acts systemically within the plant. By exposing grafted plants bearing both tolerant and sensitive shoots, the trait was functionally located in the shoot rather than the roots. Additionally, an increase in continuous-light tolerance was observed in sensitive plants when a continuous-light-tolerant shoot was grafted on it. Cultivation of greenhouse tomatoes under continuous light promises high yield increases. Our results show that to pursuit this, the trait should be bred into scion rather than rootstock lines. In addition, identifying the nature of the signal/molecule(s) and/or the mechanism of graft-induced, continuous-light tolerance can potentially result in a better understanding of important physiological processes like long-distance signaling.

A single locus confers tolerance to continuous light and allows substantial yield increase in tomato.

An important constraint for plant biomass production is the natural day length. Artificial light allows for longer photoperiods, but tomato plants develop a detrimental leaf injury when grown under continuous light--a still poorly understood phenomenon discovered in the 1920s. Here, we report a dominant locus on chromosome 7 of wild tomato species that confers continuous light tolerance. Genetic evidence, RNAseq data, silencing experiments and sequence analysis all point to the type III light harvesting chlorophyll a/b binding protein 13 (CAB-13) gene as a major factor responsible for the tolerance. In Arabidopsis thaliana, this protein is thought to have a regulatory role balancing light harvesting by photosystems I and II. Introgressing the tolerance into modern tomato hybrid lines, results in up to 20% yield increase, showing that limitations for crop productivity, caused by the adaptation of plants to the terrestrial 24-h day/night cycle, can be overcome.

Group VII ethylene response factor diversification and regulation in four species from flood-prone environments.

Flooding events negatively affect plant performance and survival. Flooding gradients thereby determine the dynamics in vegetation composition and species abundance. In adaptation to flooding, the group VII Ethylene Response Factor genes (ERF-VIIs) play pivotal roles in rice and Arabidopsis through regulation of anaerobic gene expression and antithetical survival strategies. We investigated if ERF-VIIs have a similar role in mediating survival strategies in eudicot species from flood-prone environments. Here, we studied the evolutionary origin and regulation of ERF-VII transcript abundance and the physiological responses in species from two genera of divergent taxonomic lineages (Rumex and Rorippa). Synteny analysis revealed that angiosperm ERF-VIIs arose from two ancestral loci and that subsequent diversification and duplication led to the present ERF-VII variation. We propose that subtle variation in the regulation of ERF-VII transcript abundance could explain variation in tolerance among Rorippa species. In Rumex, the main difference in flood tolerance correlated with the genetic variation in ERF-VII genes. Large transcriptional differences were found by comparing the two genera: darkness and dark submergence-induced Rumex ERF-VIIs, whereas HRE2 expression was increased in submerged Rorippa roots. We conclude that the involvement of ERF-VIIs in flooding tolerance developed in a phylogenetic-dependent manner, with subtle variations within taxonomic clades.

Plants under continuous light.

Continuous light is an essential tool for understanding the plant circadian clock. Additionally, continuous light might increase greenhouse food production. However, using continuous light in research and practice has its challenges. For instance, most of the circadian clock-oriented experiments were performed under continuous light; consequently, interactions between the circadian clock and the light signaling pathway were overlooked. Furthermore, in some plant species continuous light induces severe injury, which is only poorly understood so far. In this review paper, we aim to combine the current knowledge with a modern conceptual framework. Modern genomic tools and rediscovered continuous light-tolerant tomato species (Solanum spp.) could boost the understanding of the physiology of plants under continuous light.

Relation among plant growth, carbohydrates and flowering time in the Arabidopsis Landsberg erecta x Kondara recombinant inbred line population.

Arabidopsis thaliana natural variation was used to study plant performance viewed as the accumulation of photo-assimilates, their allocation and storage, in relation to other growth-related features and flowering-related traits. Quantitative trait locus (QTL) analysis using recombinant inbred lines derived from the cross between Landsberg erecta (originating from Poland) and Kondara (originating from Tajikistan) grown on hydroponics, revealed QTLs for the different aspects of plant growth-related traits, sugar and starch contents and flowering-related traits. Co-locations of QTLs for these different aspects were detected at different regions, mainly at the ER locus; the top of chromosomes 3, 4 and 5; and the bottom of chromosome 5. Increased plant growth was associated with early flowering and leaf transitory starch, and correlated negatively with the levels of soluble sugar at early phases of development. From the significant correlations and the co-locations of the QTLs for these aspects, we conclude that there is a complex relationship between plant growth-related traits, carbohydrate content and flowering-related traits.

What has natural variation taught us about plant development, physiology, and adaptation?

Nearly 100 genes and functional polymorphisms underlying natural variation in plant development and physiology have been identified. In crop plants, these include genes involved in domestication traits, such as those related to plant architecture, fruit and seed structure and morphology, as well as yield and quality traits improved by subsequent crop breeding. In wild plants, comparable traits have been dissected mainly in Arabidopsis thaliana. In this review, we discuss the major contributions of the analysis of natural variation to our understanding of plant development and physiology, focusing in particular on the timing of germination and flowering, plant growth and morphology, primary metabolism, and mineral accumulation. Overall, functional polymorphisms appear in all types of genes and gene regions, and they may have multiple mutational causes. However, understanding this diversity in relation to adaptation and environmental variation is a challenge for which tools are now available.