Allelic variants confer Arabidopsis adaptation to small regional environmental differences.

Natural populations of Arabidopsis thaliana provide powerful systems to study the adaptation of wild plant species. Previous research has predominantly focused on global populations or accessions collected from regions with diverse climates. However, little is known about the genetics underlying adaptation in regions with mild environmental clines. We have examined a diversity panel consisting of 192 A. thaliana accessions collected from the Netherlands, a region with limited climatic variation. Despite the relatively uniform climate, we identified evidence of local adaptation within this population. Notably, semidwarf accessions, due to mutation of the GIBBERELLIC ACID REQUIRING 5 (GA5) gene, occur at a relatively high frequency near the coast and these displayed enhanced tolerance to high wind velocities. Additionally, we evaluated the performance of the population under iron deficiency conditions and found that allelic variation in the FE SUPEROXIDE DISMUTASE 3 (FSD3) gene affects tolerance to low iron levels. Moreover, we explored patterns of local adaptation to environmental clines in temperature and precipitation, observing that allelic variation at LA RELATED PROTEIN 1C (LARP1c) likely affects drought tolerance. Not only is the genetic variation observed in a diversity panel of A. thaliana collected in a region with mild environmental clines comparable to that in collections sampled over larger geographic ranges but it is also sufficiently rich to elucidate the genetic and environmental factors underlying natural plant adaptation.

A genome-wide association study identifies novel players in Na and Fe homeostasis in Arabidopsis thaliana under alkaline-salinity stress.

In nature, multiple stress factors occur simultaneously. The screening of natural diversity panels and subsequent Genome-Wide Association Studies (GWAS) is a powerful approach to identify genetic components of various stress responses. Here, the nutritional status variation of a set of 270 natural accessions of Arabidopsis thaliana grown on a natural saline-carbonated soil is evaluated. We report significant natural variation on leaf Na (LNa) and Fe (LFe) concentrations in the studied accessions. Allelic variation in the NINJA and YUC8 genes is associated with LNa diversity, and variation in the ALA3 is associated with LFe diversity. The allelic variation detected in these three genes leads to changes in their mRNA expression and correlates with plant differential growth performance when plants are exposed to alkaline salinity treatment under hydroponic conditions. We propose that YUC8 and NINJA expression patters regulate auxin and jasmonic signaling pathways affecting plant tolerance to alkaline salinity. Finally, we describe an impairment in growth and leaf Fe acquisition associated with differences in root expression of ALA3, encoding a phospholipid translocase active in plasma membrane and the trans Golgi network which directly interacts with proteins essential for the trafficking of PIN auxin transporters, reinforcing the role of phytohormonal processes in regulating ion homeostasis under alkaline salinity.

Uncovering the physiology and distribution of thallium in Tl-hyperaccumulating and Tl-sensitive populations of Biscutella laevigata L.

BACKGROUND AND AIMS: Thallium (Tl) is extremely toxic to all lifeforms and an emerging pollutant. Plants in the Brassicaceae family, including edible crops, have an enhanced capacity for Tl accumulation, even from soils with low thallium concentration. The most extreme Tl hyperaccumulator is Biscutella laevigata, capable of attaining >32,000 µg Tl g-1 DW in its leaves. METHODS: Biscutella laevigata from a non-metallicolous accession (Feltre, Italy) and a metallicolous accession (Les Malines, France) were subjected to a dosing experiment in hydroponics (0, 5, 30 µM Tl), followed by synchrotron-based µXRF analysis to elucidate tissue and cellular-level Tl distribution. KEY RESULTS: Flow cytometric data on the two used accessions showed the Feltre accession has a genome size twice of that of the Les Malines accession (256 and 125 pg/2C respectively), suggesting they are phylogenetically distant populations. The Feltre accession does not accumulate Tl (125 µg Tl g-1 DW on average in leaves) at the 5 µM Tl dose level, whereas the Les Malines accession had a mean of 1750 µg Tl g-1 DW, with peaks of 24,130 µg Tl g-1 DW at the 30 µM Tl dose level. At 30 µM Tl the non-metallicolous accession did not grow, and at 5 µM Tl showed reduced biomasss compared to the metallicolous one. In Les Malines accession, the synchrotron-based µXRF analysis revealed that Tl is localised in the vacuoles of epidermal cells, especially underneath trichomes and in trichome basal cells. Thallium also occurs in solid crystalline deposits (3-5 µm in size, ~40 wt% Tl) that are mainly found in foliar margins and under trichome bases. CONCLUSIONS: Biscutella laevigata is an attractive model for studying Tl hypertolerance and hyperaccumulation on account of the extreme expression of this trait, and its marked intraspecific variability.

Expanding the Triangle of U: Comparative analysis of the Hirschfeldia incana genome provides insights into chromosomal evolution, phylogenomics and high photosynthesis-related traits.

BACKGROUND AND AIMS: The Brassiceae tribe encompasses many economically important crops and exhibits high intraspecific and interspecific phenotypic variation. After a shared whole-genome triplication (WGT) event (Br-α, ~15.9 million years ago), differential lineage diversification and genomic changes contributed to an array of divergence in morphology, biochemistry, and physiology underlying photosynthesis-related traits. Here, the C3 species Hirschfeldia incana is studied as it displays high photosynthetic rates under high-light conditions. Our aim was to elucidate the evolution that gave rise to the genome of H. incana and its high-photosynthesis traits. METHODS: We reconstructed a chromosome-level genome assembly for H. incana (Nijmegen, v2.0) using nanopore and chromosome conformation capture (Hi-C) technologies, with 409Mb in size and an N50 of 52Mb (a 10× improvement over the previously published scaffold-level v1.0 assembly). The updated assembly and annotation was subsequently employed to investigate the WGT history of H. incana in a comparative phylogenomic framework from the Brassiceae ancestral genomic blocks and related diploidized crops. KEY RESULTS: Hirschfeldia incana (x=7) shares extensive genome collinearity with Raphanus sativus (x=9). These two species share some commonalities with Brassica rapa and B. oleracea (A genome, x=10 and C genome, x=9, respectively) and other similarities with B. nigra (B genome, x=8). Phylogenetic analysis revealed that H. incana and R. sativus form a monophyletic clade in between the Brassica A/C and B genomes. We postulate that H. incana and R. sativus genomes are results of hybridization or introgression of the Brassica A/C and B genome types. Our results might explain the discrepancy observed in published studies regarding phylogenetic placement of H. incana and R. sativus in relation to the "Triangle of U" species. Expression analysis of WGT retained gene copies revealed sub-genome expression divergence, likely due to neo- or sub-functionalization. Finally, we highlighted genes associated with physio-biochemical-anatomical adaptive changes observed in H. incana which likely facilitate its high-photosynthesis traits under high light. CONCLUSIONS: The improved H. incana genome assembly, annotation and results presented in this work will be a valuable resource for future research to unravel the genetic basis of its ability to maintain a high photosynthetic efficiency in high-light conditions and thereby improve photosynthesis for enhanced agricultural production.

The genome sequence of Hirschfeldia incana, a new Brassicaceae model to improve photosynthetic light-use efficiency.

Photosynthesis is a key process in sustaining plant and human life. Improving the photosynthetic capacity of agricultural crops is an attractive means to increase their yields. While the core mechanisms of photosynthesis are highly conserved in C3 plants, these mechanisms are very flexible, allowing considerable diversity in photosynthetic properties. Among this diversity is the maintenance of high photosynthetic light-use efficiency at high irradiance as identified in a small number of exceptional C3 species. Hirschfeldia incana, a member of the Brassicaceae family, is such an exceptional species, and because it is easy to grow, it is an excellent model for studying the genetic and physiological basis of this trait. Here, we present a reference genome of H. incana and confirm its high photosynthetic light-use efficiency. While H. incana has the highest photosynthetic rates found so far in the Brassicaceae, the light-saturated assimilation rates of closely related Brassica rapa and Brassica nigra are also high. The H. incana genome has extensively diversified from that of B. rapa and B. nigra through large chromosomal rearrangements, species-specific transposon activity, and differential retention of duplicated genes. Duplicated genes in H. incana, B. rapa, and B. nigra that are involved in photosynthesis and/or photoprotection show a positive correlation between copy number and gene expression, providing leads into the mechanisms underlying the high photosynthetic efficiency of these species. Our work demonstrates that the H. incana genome serves as a valuable resource for studying the evolution of high photosynthetic light-use efficiency and enhancing photosynthetic rates in crop species.

NICOTIANAMINE SYNTHASE activity affects nucleolar iron accumulation and impacts rDNA silencing and RNA methylation in Arabidopsis.

In plant cells, a large pool of iron (Fe) is contained in the nucleolus, as well as in chloroplasts and mitochondria. A central determinant for intracellular distribution of Fe is nicotianamine (NA) generated by NICOTIANAMINE SYNTHASE (NAS). Here, we used Arabidopsis thaliana plants with disrupted NAS genes to study the accumulation of nucleolar iron and understand its role in nucleolar functions and more specifically in rRNA gene expression. We found that nas124 triple mutant plants, which contained lower quantities of the iron ligand NA, also contained less iron in the nucleolus. This was concurrent with the expression of normally silenced rRNA genes from nucleolar organizer regions 2 (NOR2). Notably, in nas234 triple mutant plants, which also contained lower quantities of NA, nucleolar iron and rDNA expression were not affected. In contrast, in both nas124 and nas234, specific RNA modifications were differentially regulated in a genotype dependent manner. Taken together, our results highlight the impact of specific NAS activities in RNA gene expression. We discuss the interplay between NA and nucleolar iron with rDNA functional organization and RNA methylation.

Construction and analysis of a Noccaea caerulescens TILLING population.

BACKGROUND: Metals such as Zn or Cd are toxic to plant and humans when they are exposed in high quantities through contaminated soil or food. Noccaea caerulescens, an extraordinary Zn/Cd/Ni hyperaccumulating species, is used as a model plant for metal hyperaccumulation and phytoremediation studies. Current reverse genetic techniques to generate mutants based on transgenesis is cumbersome due to the low transformation efficiency of this species. We aimed to establish a mutant library for functional genomics by a non-transgenic approach, to identify mutants with an altered mineral profiling, and to screen for mutations in bZIP19, a regulator of Zn homeostasis in N. caerulescens. RESULTS: To generate the N. caerulescens mutant library, 3000 and 5000 seeds from two sister plants of a single-seed recurrent inbred descendant of the southern French accession Saint-Félix-de-Pallières (SF) were mutagenized respectively by 0.3 or 0.4% ethyl methane sulfonate (EMS). Two subpopulations of 5000 and 7000 M2 plants were obtained after 0.3 or 0.4% EMS treatment. The 0.4% EMS treatment population had a higher mutant frequency and was used for TILLING. A High Resolution Melting curve analysis (HRM) mutation screening platform was optimized and successfully applied to detect mutations for NcbZIP19, encoding a transcription factor controlling Zn homeostasis. Of four identified point mutations in NcbZIP19, two caused non-synonymous substitutions, however, these two mutations did not alter the ionome profile compared to the wild type. Forward screening of the 0.4% EMS treatment population by mineral concentration analysis (ionomics) in leaf material of each M2 plant revealed putative mutants affected in the concentration of one or more of the 20 trace elements tested. Several of the low-Zn mutants identified in the ionomic screen did not give progeny, illustrating the importance of Zn for the species. The mutant frequency of the population was evaluated based on an average of 2.3 knockout mutants per tested monogenic locus. CONCLUSIONS: The 0.4% EMS treatment population is effectively mutagenized suitable for forward mutant screens and TILLING. Difficulties in seed production in low Zn mutants, obtained by both forward and reverse genetic approach, hampered further analysis of the nature of the low Zn phenotypes.

Micro-analytical and molecular approaches for understanding the distribution, biochemistry, and molecular biology of selenium in (hyperaccumulator) plants.

MAIN CONCLUSION: Micro-analytical techniques to untangle Se distribution and chemical speciation in plants coupled with molecular biology analysis enable the deciphering of metabolic pathways responsible for Se tolerance and accumulation. Selenium (Se) is not essential for plants and is toxic at high concentrations. However, Se hyperaccumulator plants have evolved strategies to both tolerate and accumulate > 1000 µg Se g-1 DW in their living above-ground tissues. Given the complexity of the biochemistry of Se, various approaches have been adopted to study Se metabolism in plants. These include X-ray-based techniques for assessing distribution and chemical speciation of Se, and molecular biology techniques to identify genes implicated in Se uptake, transport, and assimilation. This review presents these techniques, synthesises the current state of knowledge on Se metabolism in plants, and highlights future directions for research into Se (hyper)accumulation and tolerance. We conclude that powerful insights may be gained from coupling information on the distribution and chemical speciation of Se to genome-scale studies to identify gene functions and molecular mechanisms that underpin Se tolerance and accumulation in these ecologically and biotechnologically important plants species. The study of Se metabolism is challenging and is a useful testbed for developing novel analytical approaches that are potentially more widely applicable to the study of the regulation of a wide range of metal(loid)s in hyperaccumulator plants.

Genetics as a key to improving crop photosynthesis.

Since the basic biochemical mechanisms of photosynthesis are remarkably conserved among plant species, genetic modification approaches have so far been the main route to improve the photosynthetic performance of crops. Yet, phenotypic variation observed in wild species and between varieties of crop species implies there is standing natural genetic variation for photosynthesis, offering a largely unexplored resource to use for breeding crops with improved photosynthesis and higher yields. The reason this has not yet been explored is that the variation probably involves thousands of genes, each contributing only a little to photosynthesis, making them hard to identify without proper phenotyping and genetic tools. This is changing, though, and increasingly studies report on quantitative trait loci for photosynthetic phenotypes. So far, hardly any of these quantitative trait loci have been used in marker assisted breeding or genomic selection approaches to improve crop photosynthesis and yield, and hardly ever have the underlying causal genes been identified. We propose to take the genetics of photosynthesis to a higher level, and identify the genes and alleles nature has used for millions of years to tune photosynthesis to be in line with local environmental conditions. We will need to determine the physiological function of the genes and alleles, and design novel strategies to use this knowledge to improve crop photosynthesis through conventional plant breeding, based on readily available crop plant germplasm. In this work, we present and discuss the genetic methods needed to reveal natural genetic variation, and elaborate on how to apply this to improve crop photosynthesis.

Isotopic signatures reveal zinc cycling in the natural habitat of hyperaccumulator Dichapetalum gelonioides subspecies from Malaysian Borneo.

BACKGROUND: Some subspecies of Dichapetalum gelonioides are the only tropical woody zinc (Zn)-hyperaccumulator plants described so far and the first Zn hyperaccumulators identified to occur exclusively on non-Zn enriched 'normal' soils. The aim of this study was to investigate Zn cycling in the parent rock-soil-plant interface in the native habitats of hyperaccumulating Dichapetalum gelonioides subspecies (subsp. pilosum and subsp. sumatranum). We measured the Zn isotope ratios (δ66Zn) of Dichapetalum plant material, and associated soil and parent rock materials collected from Sabah (Malaysian Borneo). RESULTS: We found enrichment in heavy Zn isotopes in the topsoil (δ66Zn 0.13 ‰) relative to deep soil (δ66Zn -0.15 ‰) and bedrock (δ66Zn -0.90 ‰). This finding suggests that both weathering and organic matter influenced the Zn isotope pattern in the soil-plant system, with leaf litter cycling contributing significantly to enriched heavier Zn in topsoil. Within the plant, the roots were enriched in heavy Zn isotopes (δ66Zn ~ 0.60 ‰) compared to mature leaves (δ66Zn ~ 0.30 ‰), which suggests highly expressed membrane transporters in these Dichapetalum subspecies preferentially transporting lighter Zn isotopes during root-to-shoot translocation. The shoots, mature leaves and phloem tissues were enriched in heavy Zn isotopes (δ66Zn 0.34-0.70 ‰) relative to young leaves (δ66Zn 0.25 ‰). Thisindicates that phloem sources are enriched in heavy Zn isotopes relative to phloem sinks, likely because of apoplastic retention and compartmentalization in the Dichapetalum subspecies. CONCLUSIONS: The findings of this study reveal Zn cycling in the rock-soil-plant continuum within the natural habitat of Zn hyperaccumulating subspecies of Dichapetalum gelonioides from Malaysian Borneo. This study broadens our understanding of the role of a tropical woody Zn hyperaccumulator plant in local Zn cycling, and highlights the important role of leaf litter recycling in the topsoil Zn budget. Within the plant, phloem plays key role in Zn accumulation and redistribution during growth and development. This study provides an improved understanding of the fate and behaviour of Zn in hyperaccumulator soil-plant systems, and these insights may be applied in the biofortification of crops with Zn.

Converging phenomics and genomics to study natural variation in plant photosynthetic efficiency.

In recent years developments in plant phenomic approaches and facilities have gradually caught up with genomic approaches. An opportunity lies ahead to dissect complex, quantitative traits when both genotype and phenotype can be assessed at a high level of detail. This is especially true for the study of natural variation in photosynthetic efficiency, for which forward genetics studies have yielded only a little progress in our understanding of the genetic layout of the trait. High-throughput phenotyping, primarily from chlorophyll fluorescence imaging, should help to dissect the genetics of photosynthesis at the different levels of both plant physiology and development. Specific emphasis should be directed towards understanding the acclimation of the photosynthetic machinery in fluctuating environments, which may be crucial for the identification of genetic variation for relevant traits in food crops. Facilities should preferably be designed to accommodate phenotyping of photosynthesis-related traits in such environments. The use of forward genetics to study the genetic architecture of photosynthesis is likely to lead to the discovery of novel traits and/or genes that may be targeted in breeding or bio-engineering approaches to improve crop photosynthetic efficiency. In the near future, big data approaches will play a pivotal role in data processing and streamlining the phenotype-to-gene identification pipeline.

Transcriptional effects of cadmium on iron homeostasis differ in calamine accessions of Noccaea caerulescens.

Calamine accessions of the zinc/cadmium/nickel hyperaccumulator, Noccaea caerulescens, exhibit striking variation in foliar cadmium accumulation in nature. The Ganges accession (GA) from Southern France displays foliar cadmium hyperaccumulation (>1000 µg g-1 DW), whereas the accession La Calamine (LC) from Belgium, with similar local soil metal composition, does not (<100 µg g-1 DW). All calamine accessions are cadmium hypertolerant. To find out the differences between LC and GA in their basic adaptation mechanisms, we bypassed the cadmium excluding phenotype of LC by exposing the plants to 50 µm cadmium in hydroponics, achieving equal cadmium accumulation in the shoots. The iron content increased in the roots of both accessions. GA exhibited significant decreases in manganese and zinc contents in the roots and shoots, approaching those in LC. Altogether 702 genes responded differently to cadmium exposure between the accessions, 157 and 545 in the roots and shoots, respectively. Cadmium-exposed LC showed a stress response and had decreased levels of a wide range of photosynthesis-related transcripts. GA showed less changes, mainly exhibiting an iron deficiency-like response. This included increased expression of genes encoding five iron deficiency-regulated bHLH transcription factors, ferric reduction oxidase FRO2, iron transporters IRT1 and OPT3, and nicotianamine synthase NAS1, and decreased expression of genes encoding ferritins and NEET (a NEET family iron-sulfur protein), which is possibly involved in iron transfer, distribution and/or management. The function of the IRT1 gene in the accessions was compared. We conclude that the major difference between the two accessions is in the way they cope with iron under cadmium exposure.

Natural variation of photosynthetic efficiency in Arabidopsis thaliana accessions under low temperature conditions.

Low, but non-freezing, temperatures have negative effects on plant growth and development. Despite some molecular signalling pathways being known, the mechanisms causing different responses among genotypes are still poorly understood. Photosynthesis is one of the processes that are affected by low temperatures. Using an automated phenotyping platform for chlorophyll fluorescence imaging the steady state quantum yield of photosystem II (PSII) electron transport (ΦPSII ) was measured and used to quantify the effect of moderately low temperature on a population of Arabidopsis thaliana natural accessions. Observations were made over the course of several weeks in standard and low temperature conditions and a strong decrease in ΦPSII upon the cold treatment was found. A genome wide association study identified several quantitative trait loci (QTLs) that are associated with changes in ΦPSII in low temperature. One candidate for a cold specific QTL was validated with a mutant analysis to be one of the genes that is likely involved in the PSII response to the cold treatment. The gene encodes the PSII associated protein PSB27 which has already been implicated in the adaptation to fluctuating light.

Rice F-bZIP transcription factors regulate the zinc deficiency response.

The F-bZIP transcription factors bZIP19 and bZIP23 are the central regulators of the zinc deficiency response in Arabidopsis, and phylogenetic analysis of F-bZIP homologs across land plants indicates that the regulatory mechanism of the zinc deficiency response may be conserved. Here, we identified the rice F-bZIP homologs and investigated their function. OsbZIP48 and OsbZIP50, but not OsbZIP49, complement the zinc deficiency-hypersensitive Arabidopsis bzip19bzip23 double mutant. Ectopic expression of OsbZIP50 in Arabidopsis significantly increases plant zinc accumulation under control zinc supply, suggesting an altered Zn sensing in OsbZIP50. In addition, we performed a phylogenetic analysis of F-bZIP homologs from representative monocot species that supports the branching of plant F-bZIPs into Group 1 and Group 2. Our results suggest that regulation of the zinc deficiency response in rice is conserved, with OsbZIP48 being a functional homolog of AtbZIP19 and AtbZIP23. A better understanding of the mechanisms behind the Zn deficiency response in rice and other important crops will contribute to develop plant-based strategies to address the problems of Zn deficiency in soils, crops, and cereal-based human diets.

Overexpression of the MYB29 transcription factor affects aliphatic glucosinolate synthesis in Brassica oleracea.

KEY MESSAGE: Overexpression of BoMYB29 gene up-regulates the aliphatic glucosinolate pathway in Brassica oleracea plants increasing the production of the anti-cancer metabolite glucoraphanin, and the toxic and pungent sinigrin. Isothiocyanates, the bio-active hydrolysis products of glucosinolates, naturally produced by several Brassicaceae species, play an important role in human health and agriculture. This study aims at correlating the content of aliphatic glucosinolates to the expression of genes involved in their synthesis in Brassica oleracea, and perform functional analysis of BoMYB29 gene. To this purpose, three genotypes were used: a sprouting broccoli, a cabbage, and a wild genotype (Winspit), a high glucosinolate containing accession. Winspit showed the highest transcript level of BoMYB28, BoMYB29 and BoAOP2 genes, and BoAOP2 expression was positively correlated with that of the two MYB genes. Further analyses of the aliphatic glucosinolates also showed a positive correlation between the expression of BoAOP2 and the production of sinigrin and gluconapin in Winspit. The Winspit BoMYB29 CDS was cloned and overexpressed in Winspit and in the DH AG1012 line. Overexpressing Winspit plants produced higher quantities of alkenyl glucosinolates, such as sinigrin. Conversely, the DH AG1012 transformants showed a higher production of methylsulphinylalkyl glucosinolates, including glucoraphanin, and, despite an up-regulation of the aliphatic glucosinolate genes, no increase in alkenyl glucosinolates. The latter may be explained by the absence of a functional AOP2 gene in DH AG1012. Nevertheless, an extract of DH AG1012 lines overexpressing BoMYB29 provided a chemoprotective effect on human colon cells. This work exemplifies how the genetic diversity of B. oleracea may be used by breeders to select for higher expression of transcription factors for glucosinolate biosynthesis to improve its natural, health-promoting properties.

QTL and candidate genes associated with leaf anion concentrations in response to phosphate supply in Arabidopsis thaliana.

BACKGROUND: Phosphorus is often present naturally in the soil as inorganic phosphate, Pi, which bio-availability is limited in many ecosystems due to low soil solubility and mobility. Plants respond to low Pi with a Pi Starvation Response, involving Pi sensing and long-distance signalling. There is extensive cross-talk between Pi homeostasis mechanisms and the homeostasis mechanism for other anions in response to Pi availability. RESULTS: Recombinant Inbred Line (RIL) and Genome Wide Association (GWA) mapping populations, derived from or composed of natural accessions of Arabidopsis thaliana, were grown under sufficient and deficient Pi supply. Significant treatment effects were found for all traits and significant genotype x treatment interactions for the leaf Pi and sulphate concentrations. Using the RIL/QTL population, we identified 24 QTLs for leaf concentrations of Pi and other anions, including a major QTL for leaf sulphate concentration (SUL2) mapped to the bottom of chromosome (Chr) 1. GWA mapping found 188 SNPs to be associated with the measured traits, corresponding to 152 genes. One of these SNPs, associated with leaf Pi concentration, mapped to PP2A-1, a gene encoding an isoform of the catalytic subunit of a protein phosphatase 2A. Of two additional SNPs, associated with phosphate use efficiency (PUE), one mapped to AT5G49780, encoding a leucine-rich repeat protein kinase involved in signal transduction, and the other to SIZ1, a gene encoding a SUMO E3 ligase, and a known regulator of P starvation-dependent responses. One SNP associated with leaf sulphate concentration was found in SULTR2;1, encoding a sulphate transporter, known to enhance sulphate translocation from root to shoot under P deficiency. Finally, one SNP was mapped to FMO GS-OX4, a gene encoding glucosinolate S-oxygenase involved in glucosinolate biosynthesis, which located within the confidence interval of the SUL2 locus. CONCLUSION: We identified several candidate genes with known functions related to anion homeostasis in response to Pi availability. Further molecular studies are needed to confirm and validate these candidate genes and understand their roles in examined traits. Such knowledge will contribute to future breeding for improved crop PUE .

Genomics of Adaptation Depends on the Rate of Environmental Change in Experimental Yeast Populations.

The rate of directional environmental change may have profound consequences for evolutionary dynamics and outcomes. Yet, most evolution experiments impose a sudden large change in the environment, after which the environment is kept constant. We previously cultured replicate Saccharomyces cerevisiae populations for 500 generations in the presence of either gradually increasing or constant high concentrations of the heavy metals cadmium, nickel, and zinc. Here, we investigate how each of these treatments affected genomic evolution. Whole-genome sequencing of evolved clones revealed that adaptation occurred via a combination of SNPs, small indels, and whole-genome duplications and other large-scale structural changes. In contrast to some theoretical predictions, gradual and abrupt environmental change caused similar numbers of genomic changes. For cadmium, which is toxic already at comparatively low concentrations, mutations in the same genes were used for adaptation to both gradual and abrupt increase in concentration. Conversely, for nickel and zinc, which are toxic at high concentrations only, mutations in different genes were used for adaptation depending on the rate of change. Moreover, evolution was more repeatable following a sudden change in the environment, particularly for nickel and zinc. Our results show that the rate of environmental change and the nature of the selection pressure are important drivers of evolutionary dynamics and outcomes, which has implications for a better understanding of societal problems such as climate change and pollution.

Transcriptome dynamics of Arabidopsis during sequential biotic and abiotic stresses.

In nature, plants have to cope with a wide range of stress conditions that often occur simultaneously or in sequence. To investigate how plants cope with multi-stress conditions, we analyzed the dynamics of whole-transcriptome profiles of Arabidopsis thaliana exposed to six sequential double stresses inflicted by combinations of: (i) infection by the necrotrophic fungus Botrytis cinerea, (ii) herbivory by chewing larvae of Pieris rapae, and (iii) drought stress. Each of these stresses induced specific expression profiles over time, in which one-third of all differentially expressed genes was shared by at least two single stresses. Of these, 394 genes were differentially expressed during all three stress conditions, albeit often in opposite directions. When two stresses were applied in sequence, plants displayed transcriptome profiles that were very similar to the second stress, irrespective of the nature of the first stress. Nevertheless, significant first-stress signatures could be identified in the sequential stress profiles. Bioinformatic analysis of the dynamics of co-expressed gene clusters highlighted specific clusters and biological processes of which the timing of activation or repression was altered by a prior stress. The first-stress signatures in second stress transcriptional profiles were remarkably often related to responses to phytohormones, strengthening the notion that hormones are global modulators of interactions between different types of stress. Because prior stresses can affect the level of tolerance against a subsequent stress (e.g. prior herbivory strongly affected resistance to B. cinerea), the first-stress signatures can provide important leads for the identification of molecular players that are decisive in the interactions between stress response pathways.

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.

Multi-Element Bioimaging of Arabidopsis thaliana Roots.

Better understanding of root function is central for the development of plants with more efficient nutrient uptake and translocation. We here present a method for multielement bioimaging at the cellular level in roots of the genetic model system Arabidopsis (Arabidopsis thaliana). Using conventional protocols for microscopy, we observed that diffusible ions such as potassium and sodium were lost during sample dehydration. Thus, we developed a protocol that preserves ions in their native, cellular environment. Briefly, fresh roots are encapsulated in paraffin, cryo-sectioned, and freeze dried. Samples are finally analyzed by laser ablation-inductively coupled plasma-mass spectrometry, utilizing a specially designed internal standard procedure. The method can be further developed to maintain the native composition of proteins, enzymes, RNA, and DNA, making it attractive in combination with other omics techniques. To demonstrate the potential of the method, we analyzed a mutant of Arabidopsis unable to synthesize the metal chelator nicotianamine. The mutant accumulated substantially more zinc and manganese than the wild type in the tissues surrounding the vascular cylinder. For iron, the images looked completely different, with iron bound mainly in the epidermis of the wild-type plants but confined to the cortical cell walls of the mutant. The method offers the power of inductively coupled plasma-mass spectrometry to be fully employed, thereby providing a basis for detailed studies of ion transport in roots. Being applicable to Arabidopsis, the molecular and genetic approaches available in this system can now be fully exploited in order to gain a better mechanistic understanding of these processes.