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Altres ajuts: acords transformatius de la UAB 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.

Background and aims: Phytoextraction is an eco-friendly approach for remediation of heavy metal contaminated soil. The aim is to screen Noccaea caerulescens lines with higher cadmium (Cd) phytoextraction efficiency and investigate differences in Cd species and distribution in the leaves of high and low Cd accumulating lines. Methods: Biomass production and Cd bioaccumulation capacities of 29 Noccaea caerulescens lines, generated through single-seed-descent from a Cd hyperaccumulating calamine population, were assessed in a pot experiment with a moderately Cd contaminated soil (2.1 mg Cd kg− 1). Synchrotron-based techniques were employed to identify and characterize Cd speciation and distribution in Noccaea caerulescens leaves. Results: The largest biomass of Noccaea caerulescens reached 5.0 ± 3.3 g (D. W. pot− 1) after 6 months growth. The Cd concentrations in shoots varied from 85 to 203 mg kg− 1. The most efficient line removed 0.64 mg Cd pot− 1 and lowered the total Cd in soil by 30%. Synchrotron-based X-ray absorption spectroscopy showed that the dominant Cd species was Cd-thiol complexes. Cadmium-carboxyl and Cd-phytate/phosphate were present in the leaves of high and low Cd accumulating lines, respectively. Micro X-ray fluorescence microscopy showed cadmium was concentrated in leaf veins. Conclusions: There are wide variations including both biomass production and Cd accumulation capacity among different lines within the same calamine ecotype of Noccaea caerulescens. Cadmium-thiol complexes play the most important role in Cd detoxification in leaves of Noccaea caerulescens grown in moderately Cd contaminated paddy soil. These findings provide a physiological basis for breeding high Cd accumulation varieties of Noccaea caerulescens.

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. Abstract 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. Graphical Abstract

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.

Aims: The study aimed at characterizing the patterns of natural variation in the tolerance and accumulation capacities for zinc (Zn), cadmium (Cd), and nickel (Ni) between and within edaphic ecotypes of the Zn/Cd/Ni hyperaccumulator, Noccaea caerulescens. Methods: Tolerance was assessed in a hydroponic ‘sequential exposure’ test, using the lowest concentration that completely arrested root growth as an end point. Accumulation was measured as the foliar metal concentration after six weeks of growth at 5 µM Zn, 2 µM Cd, or 1 µM Ni. Results: Zn and Cd tolerance were positively correlated, and highest in the calamine ecotype. Ni tolerance was without significant ecotypic variation. The ultramafic ecotype was as Zn-tolerant as the non-metallicolous one, but much more sensitive to Cd. The accumulation capacities for Zn, Cd and Ni were all positively correlated and without significant ecotypic variation. Zn hyperaccumulation capacity was species-wide, but Cd and Ni hyperaccumulation capacities were lacking in four populations (all calamine). Conclusions: There is considerable independent variation among populations regarding their Zn, Cd, and Ni accumulation capacities. This variation is most pronounced within the calamine ecotype, because some populations apparently had adopted an exclusion strategy for Zn or Cd hypertolerance, whereas others had not.

Zinc deficiency in soils and crops is a global problem. We combine functional and phylogenetic analyses to unravel the role of rice F-bZIP homologs in 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.

Photosynthesis is a biophysical and biochemical process that plays a key role in sustaining plant and human life, being the first step in the production of energy-rich molecules and oxygen in the biosphere. Improving the photosynthetic capacity of agricultural crops is highly desirable to increase their yields. While the core mechanisms of photosynthesis are highly conserved, certainly in higher plants, plants that can maintain a high photosynthetic light-use efficiency at high irradiance are exceptional and may be useful to understand and improve high irradiance photosynthesis of crops. One such exceptional species is Hirschfeldia incana, a member of the well-studied Brassicaceae family that is easy to grow under standard laboratory conditions, providing an excellent resource for studying the genetic and physiological basis of this trait. Here, we present a reference assembly of H. incana and affirm its high photosynthetic efficiency relative to the Brassicaceae species Brassica rapa, Brassica nigra, and Arabidopsis thaliana. We estimate that it diverged from B. rapa and B. nigra 10-11 million years ago and that its genome has diversified from that of the latter two species through large chromosomal rearrangements, species-specific transposon activity, and differential retention of duplicated genes. Genes present at copy numbers different from B. rapa and B. nigra include those involved in photosynthesis and/or abiotic stress, which may mediate the high photosynthetic efficiency of H. incana. We expect the reference assembly of H. incana to be a valuable genomic resource for identifying ways to enhance photosynthetic rates in crop species.

The molecular biology and genetics of the Ni–Cd–Zn hyperaccumulator Noccaea caerulescens has been extensively studied, but no information is yet available on Ni and Zn redistribution and mobilization during seed germination. Due to the different physiological functions of these elements, and their associated transporter pathways, we expected differential tissue distribution and different modes of translocation of Ni and Zn during germination. This study used synchrotron X-ray fluorescence tomography techniques as well as planar elemental X-ray imaging to elucidate elemental (re)distribution at various stages of the germination process in contrasting accessions of N. caerulescens. The results show that Ni and Zn are both located primarily in the cotyledons of the emerging seedlings and Ni is highest in the ultramafic accessions (up to 0.15 wt%), whereas Zn is highest in the calamine accession (up to 600 μg g–1). The distribution of Ni and Zn in seeds was very similar, and neither element was translocated during germination. The Fe maps were especially useful to obtain spatial reference within the seeds, as it clearly marked the vasculature. This study shows how a multimodal combination of synchrotron techniques can be used to obtain powerful insights about the metal distribution in physically intact seeds and seedlings.

BackgroundHyperaccumulation of trace elements is a rare trait among plants which is being investigated to advance our understanding of the regulation of metal accumulation and applications in phytotechnologies.Noccaea caerulescens(Brassicaceae) is an intensively studied hyperaccumulator model plant capable of attaining extremely high tissue concentrations of zinc and nickel with substantial genetic variation at the population-level. Micro-X-ray Fluorescence spectroscopy (µXRF) mapping is a sensitive high-resolution technique to obtain information of the spatial distribution of the plant metallome in hydrated samples. We used laboratory-based µXRF to characterize a collection of 86 genetically diverseNoccaea caerulescensaccessions from across Europe. We developed an image-processing method to segment different plant substructures in the µXRF images. We introduced the concentration quotient (CQ) to quantify spatial patterns of metal accumulation and linked that to genetic variation.ResultsImage processing resulted in automated segmentation of µXRF plant images into petiole, leaf margin, leaf interveinal and leaf vasculature substructures. The harmonic means of recall and precision (F1 score) were 0.79, 0.80, 0.67, and 0.68, respectively. Spatial metal accumulation as determined by CQ is highly heritable inNoccaea caerulescensfor all substructures, with broad-sense heritability (H2) ranging from 76 to 92%, and correlates only weakly with other heritable traits. Insertion of noise into the image segmentation algorithm barely decreases heritability scores of CQ for the segmented substructures, illustrating the robustness of the trait and the quantification method. Very low heritability was found for CQ if randomly generated substructures were compared, validating the approach.ConclusionsA strategy for segmenting µXRF images ofNoccaea caerulescensis proposed and the concentration quotient is developed to provide a quantitative measure of metal accumulation pattern, which can be used to determine genetic variation for such pattern. The metric is robust to segmentation error and provides reliable H2estimates. This strategy provides an avenue for quantifying XRF data for analysis of the genetics of metal distribution patterns in plants and the subsequent discovery of new genes that regulate metal homeostasis and sequestration in plants.

Zinc (Zn) is an essential micronutrient for plants and animals owing to its structural and catalytic roles in many proteins

The fitness landscape is a concept that is widely used for understanding and predicting evolutionary adaptation. The topography of the fitness landscape depends critically on the environment, with potentially far-reaching consequences for evolution under changing conditions. However, few studies have assessed directly how empirical fitness landscapes change across conditions, or validated the predicted consequences of such change. We previously evolved replicate yeast populations in the presence of either gradually increasing, or constant high, concentrations of the heavy metals cadmium (Cd), nickel (Ni), and zinc (Zn), and analyzed their phenotypic and genomic changes. Here, we reconstructed the local fitness landscapes underlying adaptation to each metal by deleting all repeatedly mutated genes both by themselves and in combination. Fitness assays revealed that the height, and/or shape, of each local fitness landscape changed considerably across metal concentrations, with distinct qualitative differences between unconditionally (Cd) and conditionally toxic metals (Ni and Zn). This change in topography had particularly crucial consequences in the case of Ni, where a substantial part of the individual mutational fitness effects changed in sign across concentrations. Based on the Ni landscape analyses, we made several predictions about which mutations had been selected when during the evolution experiment. Deep sequencing of population samples from different time points generally confirmed these predictions, demonstrating the power of landscape reconstruction analyses for understanding and ultimately predicting evolutionary dynamics, even under complex scenarios of environmental change.

Cadmium (Cd) was shown to co-localise with calcium (Ca) in oxalate crystals in the stems and leaves of Cd tolerant Gomphrena claussenii, but Cd binding remained unresolved. Using synchrotron radiation X-ray absorption near edge spectroscopy we demonstrate that in oxalate crystals of hydroponically grown G. claussenii the vast majority of Cd is bound to oxygen ligands in oxalate crystals (>88%; Cd-O-C coordination) and the remaining Cd is bound to sulphur ligands (Cd-S-C coordination). Cadmium binding to oxalate does not depend on the amount of Ca supplied or from which organs the crystals originate (stems and mature leaves). By contrast, roots contain no oxalate crystals and therein Cd is bound predominantly by S ligands. The potential to remove Cd by extraction of Cd-rich oxalate crystals from plant material should be tested in phytoextraction or phytomining strategies.

Summary 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., Significance Statement The fields of genomics and phenomics in plant science have gone through a major revolution that has led to the technological and methodological maturation of both fields of study in the past decade. Expansion of high‐throughput phenotyping technology will soon allow the scaling up of forward genetics approaches in order to adequately study the genetic architecture of traits that may accelerate the much needed improvement of photosynthesis in crop plants.

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.

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., We performed a quantitative genetics analysis on an A. thaliana diversity panel to identify genetic factors involved in photosynthetic cold response. Many QTLs were identified and we present the PSB27 gene, encoding a protein associated with photosystem II repair, as a candidate for one of the QTLs.

Assessing the impact of variation in chloroplast and mitochondrial DNA (collectively termed the plasmotype) on plant phenotypes is challenging due to the difficulty in separating their effect from nuclear derived variation (the nucleotype). Haploid inducer lines can be used as efficient plasmotype donors to generate new plasmotype-nucleotype combinations (cybrids)(Ravi et al., 2014). We generated a panel comprising all possible cybrids of seven Arabidopsis thaliana accessions and extensively phenotyped these lines for 1859 phenotypes under stable and fluctuating conditions. We show that natural variation in the plasmotype results in additive as well as epistatic effects across all phenotypic categories. Plasmotypes which induce more additive phenotypic changes also cause more significant epistatic effects, suggesting a possible common basis for both additive and epistatic effects. On average epistatic interactions explained twice as much of the variance in phenotypes as additive plasmotype effects. The impact of plasmotypic variation was also more pronounced under fluctuating and stressful environmental conditions. Thus, the phenotypic impact of variation in plasmotypes is the outcome of multilevel Nucleotype X Plasmotype X Environment interactions and, as such, the plasmotype is likely to serve as a reservoir of variation which is only exposed under certain conditions. The production of cybrids using haploid inducers is a quick and precise method for assessing the phenotypic effects of natural variation in organellar genomes. It will facilitate efficient screening of unique nucleotype-plasmotype combinations to both improve our understanding of natural variation in nucleotype plasmotype interactions and identify favourable combinations to improve plant performance.

The appropriate timing of flowering is crucial for plant reproductive success. Studies of the molecular mechanism of flower induction in the model plant Arabidopsis thaliana showed long days and vernalization as major environmental promotive factors. Noccaea caerulescens has an obligate vernalization requirement that has not been studied at the molecular genetics level. Here, we characterize the vernalization requirement and response of four geographically diverse biennial/perennial N. caerulescens accessions: Ganges (GA), Lellingen (LE), La Calamine (LC), and St. Felix de Pallières (SF). Differences in vernalization responsiveness among accessions suggest that natural variation for this trait exists within N. caerulescens. Mutants which fully abolish the vernalization requirement were identified and were shown to contain mutations in the FLOWERING LOCUS C (NcFLC) and SHORT VEGETATIVE PHASE (NcSVP) genes, two key floral repressors in this species. At high temperatures, the non-vernalization requiring flc-1 mutant reverts from flowering to vegetative growth, which is accompanied with a reduced expression of LFY and AP1. This suggested there is “crosstalk” between vernalization and ambient temperature, which might be a strategy to cope with fluctuations in temperature or adopt a more perennial flowering attitude and thus facilitate a flexible evolutionary response to the changing environment across the species range.

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. Abstract 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. Electronic supplementary material The online version of this article (10.1007/s11103-019-00890-2) contains supplementary material, which is available to authorized users.

Zinc (Zn) is an essential nutrient for plants, with a crucial role as a cofactor for many enzymes. Approximately one-third of the global arable land area is Zn deficient, leading to reduced crop yield and quality. To improve crop tolerance to Zn deficiency, it is important to understand the mechanisms plants have adopted to tolerate suboptimal Zn supply. In this study, physiological and molecular aspects of traits related to Zn deficiency tolerance were examined in a panel of 19 Arabidopsis thaliana accessions. Accessions showed a larger variation for shoot biomass than for Zn concentration, indicating that they have different requirements for their minimal Zn concentration required for growth. Accessions with a higher tolerance to Zn deficiency showed an increased expression of the Zn deficiency-responsive genes ZIP4 and IRT3 in comparison with Zn deficiency-sensitive accessions. Changes in the shoot ionome, as a result of the Zn treatment of the plants, were used to build a multinomial logistic regression model able to distinguish plants regarding their Zn nutritional status. This set of biomarkers, reflecting the A. thaliana response to Zn deficiency and Zn deficiency tolerance, can be useful for future studies aiming to improve the performance and Zn status of crop plants grown under suboptimal Zn concentrations.

BackgroundPhosphorus 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.ResultsRecombinant Inbred Line (RIL) and Genome Wide Association (GWA) mapping populations, derived from or composed of natural accessions ofArabidopsis 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 toPP2A-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 toSIZ1, 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 inSULTR2;1,encoding a sulphate transporter, known to enhance sulphate translocation from root to shoot under P deficiency. Finally, one SNP was mapped toFMO GS-OX4, a gene encoding glucosinolate S-oxygenase involved in glucosinolate biosynthesis, which located within the confidence interval of theSUL2locus.ConclusionWe 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 .

Hyperaccumulator plants present the ideal model system for studying the physiological regulation of the essential (and potentially toxic) transition elements nickel and zinc. This study used synchrotron X-ray Fluorescence Microscopy (XFM) elemental imaging and spatially resolved X-ray Absorption Spectroscopy (XAS) to elucidate elemental localization and chemical speciation of nickel and zinc in the hyperaccumulators Noccaea tymphaea and Bornmuellera emarginata (synonym Leptoplax emarginata). The results show that in the leaves of N. tymphaea nickel and zinc have contrasting localization, and whereas nickel is present in vacuoles of epidermal cells, zinc occurs mainly in the mesophyll cells. In the seeds Ni and Zn are similarly localized and strongly enriched in the cotyledons in N. tymphaea. Nickel is strongly enriched in the tip of the radicle of B. emarginata. Noccaea tymphaea has an Fe-rich provascular strand network in the cotyledons of the seed. The chemical speciation of Ni in the seeds of N. tymphaea is unequivocally associated with carboxylic acids, whereas Zn is present as the phytate complex. The spatially resolved spectroscopy did not reveal any spatial variation in chemical speciation of Ni and Zn within the N. tymphaea seed. The dissimilar ecophysiological behaviour of Ni and Zn in N. tymphaea and B. emarginata raises questions about the evolution of hyperaccumulation in these species.

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 (−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.

Summary Gomphrena claussenii is a recently described zinc (Zn)- and cadmium (Cd)-hypertolerant Amaranthaceae species displaying a metal bioindicator Zn/Cd accumulation response. We investigated the Zn and Cd distribution in stem and leaf tissues of G. claussenii at the cellular level, and determined metabolite profiles to investigate metabolite involvement in Zn and Cd sequestration. Gomphrena claussenii plants exposed to high Zn and Cd supply were analysed by scanning electron microscopy with energy-dispersive X-ray (SEM-EDX) and micro-proton-induced X-ray emission (micro-PIXE). In addition, gas chromatography-time of flight-mass spectrometry (GC-TOF-MS) was used to determine metabolite profiles on high Zn and Cd exposure. Stem and leaf tissues of G. claussenii plants exposed to control and high Cd conditions showed the abundant presence of calcium oxalate (CaOx) crystals, but on high Zn exposure, their abundance was strongly reduced. Ca and Cd co-localized to the CaOx crystals in Cd-exposed plants. Citrate, malate and oxalate levels were all higher in shoot tissues of metal-exposed plants, with oxalate levels induced 2.6-fold on Zn exposure and 6.4-fold on Cd exposure. Sequestration of Cd in vacuolar CaOx crystals of G. claussenii is found to be a novel mechanism to deal with Cd accumulation and tolerance.

Aims: This study explores the biofumigation effects of glucosinolate (GSL) containing Brassica oleracea plant material on beneficial, non-target soil organisms, and aims to relate those effects to differences in GSL profiles. Methods: Leaf material of purple sprouting broccoli 'Santee', Savoy cabbage 'Wintessa', and the wild B. oleracea accession Winspit was analysed for GSL production and used for biofumigation experiments on the beneficial soil invertebrates, Folsomia candida (springtail) and Eisenia andrei (earthworm) and the soil bacterial community. Results: When mixed into soil, the Winspit plant material exerted the highest toxic effects on beneficial soil invertebrates by reducing survival and reproduction. Total GSL levels varied substantially between genotypes, in particular the aliphatic GSL (AGSL) sinigrin and gluconapin being highly abundant or exclusively present in Winspit. Differences between the genotypes regarding biofumigation effects on the soil microbial community were only observed on a temporal basis with the largest difference in bacterial community structure after 1 week. Conclusions: The high total GSL content in biofumigated soil could explain the toxicity of Winspit for soil invertebrates. These effects are likely to be the results of high AGSL levels in Winspit. The use of wild B. oleracea crops, such as Winspit, for biofumigation practices would need a proper assessment of the overall impact on soil biota before being applied on a wide scale.

All living organisms require a variety of essential elements for their basic biological functions. While the homeostasis of nutrients is highly intertwined, the molecular and genetic mechanisms of these dependencies remain poorly understood. Here, we report a discovery of a molecular pathway that controls phosphate (Pi) accumulation in plants under Zn deficiency. Using genome-wide association studies, we first identified allelic variation of the Lyso-PhosphatidylCholine (PC) AcylTransferase 1 (LPCAT1) gene as the key determinant of shoot Pi accumulation under Zn deficiency. We then show that regulatory variation at the LPCAT1 locus contributes significantly to this natural variation and we further demonstrate that the regulation of LPCAT1 expression involves bZIP23 TF, for which we identified a new binding site sequence. Finally, we show that in Zn deficient conditions loss of function of LPCAT1 increases the phospholipid Lyso-PhosphatidylCholine/PhosphatidylCholine ratio, the expression of the Pi transporter PHT1;1, and that this leads to shoot Pi accumulation.

Plants have evolved several mechanisms for sensing increased irradiance, involving signal perception by photoreceptors (cryptochromes), and subsequent biochemical (reactive oxygen species, ROS) and metabolic clues to transmit the signals. This results in the increased expression of heat‐shock response genes and of the transcription factor LONG HYPOCOTYL 5 (HY5, mediated by the cryptochrome photoreceptor 1, CRY1). Here, we show the existence of another response pathway in Arabidopsis. This pathway evokes the SPX1‐mediated expression activation of the transcription factor PHR1 and leads to the expression of several galactolipid biosynthesis genes. Gene expression analysis of accessions Col‐0, Ga‐0, and Ts‐1, showed activated expression of the SPX1/PHR1‐mediated gene expression activation pathway acting on galactolipids biosynthesis genes in both Ga‐0 and Col‐0, but not in Ts‐1. The activation of the SPX1/PHR1‐mediated response pathway can be associated with lower photosynthesis efficiency in Ts‐1, compared to Col‐0 and Ga‐0. Besides the accession‐associated activation of the SPX1/PHR1‐mediated response pathway, comparing gene expression in the accessions showed stronger activation of several heat responsive genes in Ga‐0, and the opposite in Ts‐1, when compared to Col‐0, in line with the differences in their efficiency of photosynthesis. We conclude that natural variation in activation of both heat responsive genes and of galactolipids biosynthesis genes contribute to the variation in photosynthesis efficiency in response to irradiance increase.

Zinc (Zn) and iron (Fe) deficiency are widespread among citrus plants, but the molecular mechanisms regarding uptake and transport of these two essential metal ions in citrus are still unclear. In the present study, 12 members of the Zn/Fe-regulated transporter (ZRT/IRT)-related protein (ZIP) gene family were identified and isolated from a widely used citrus rootstock, trifoliate orange (Poncirus trifoliata L. Raf.), and the genes were correspondingly named as PtZIPs according to the sequence and functional similarity to Arabidopsis thaliana ZIPs. The 12 PtZIP genes were predicted to encode proteins of 334–419 amino acids, harboring 6–9 putative transmembrane (TM) domains. All of the PtZIP proteins contained the highly conserved ZIP signature sequences in TM-IV, and nine of them showed a variable region rich in histidine residues between TM-III and TM-IV. Phylogenetic analysis subdivided the PtZIPs into four groups, similar as found for the ZIP family of A. thaliana, with clustered PtZIPs sharing a similar gene structure. Expression analysis showed that the PtZIP genes were very differently induced in roots and leaves under conditions of Zn, Fe and Mn deficiency. Yeast complementation tests indicated that PtIRT1, PtZIP1, PtZIP2, PtZIP3, and PtZIP12 were able to complement the zrt1zrt2 mutant, which was deficient in Zn uptake; PtIRT1 and PtZIP7 were able to complement the fet3fet4 mutant, which was deficient in Fe uptake, and PtIRT1 was able to complement the smf1 mutant, which was deficient in Mn uptake, suggesting their respective functions in Zn, Fe, and Mn transport. The present study broadens our understanding of metal ion uptake and transport and functional divergence of the various PtZIP genes in citrus plants.

Drought stress was imposed on two sets of Arabidopsis thaliana genotypes grown in sand under short-day conditions and analysed for several shoot and root growth traits. The response to drought was assessed for quantitative trait locus (QTL) mapping in a genetically diverse set of Arabidopsis accessions using genome-wide association (GWA) mapping, and conventional linkage analysis of a recombinant inbred line (RIL) population. Results showed significant genotype by environment interaction (G×E) for all traits in response to different watering regimes. For the RIL population, the observed G×E was reflected in 17 QTL by environment interactions (Q×E), while 17 additional QTLs were mapped not showing Q×E. GWA mapping identified 58 single nucleotide polymorphism (SNPs) associated with loci displaying Q×E and an additional 16 SNPs associated with loci not showing Q×E. Many candidate genes potentially underlying these loci were suggested. The genes for RPS3C and YLS7 were found to contain conserved amino acid differences when comparing Arabidopsis accessions with strongly contrasting drought response phenotypes, further supporting their candidacy. One of these candidate genes co-located with a QTL mapped in the RIL population.

Summary Genes underlying environmental adaptability tend to be over-retained in polyploid plant species. Zinc deficiency (ZnD) and iron deficiency (FeD), excess Zn (ZnE) and cadmium exposure (CdE) are major environmental problems for crop cultivation, but little is known about the differential expression of duplicated genes upon these stress conditions. Applying Tag-Seq technology to leaves of Brassica rapa grown under FeD, ZnD, ZnE or CdE conditions, with normal conditions as a control, we examined global gene expression changes and compared the expression patterns of multiple paralogs. We identified 812, 543, 331 and 447 differentially expressed genes under FeD, ZnD, ZnE and CdE conditions, respectively, in B. rapa leaves. Genes involved in regulatory networks centered on the transcription factors bHLH038 or bHLH100 were differentially expressed under (ZnE-induced) FeD. Further analysis revealed that genes associated with Zn, Fe and Cd responses tended to be over-retained in the B. rapa genome. Most of these multiple-copy genes showed the same direction of expression change under stress conditions. We conclude that the duplicated genes involved in trace element responses in B. rapa are functionally redundant, making the regulatory network more complex in B. rapa than in Arabidopsis thaliana.

Exploiting genetic variation for more efficient photosynthesis is an underexplored route towards new crop varieties. This study demonstrates the genetic dissection of higher plant photosynthesis efficiency down to the genomic DNA level, by confirming that allelic sequence variation at the Arabidopsis thaliana YELLOW SEEDLING1 (YS1) gene explains natural diversity in photosynthesis acclimation to high irradiance. We use a genome-wide association study to identify quantitative trait loci (QTLs) involved in the Arabidopsis photosynthetic acclimation response. Candidate genes underlying the QTLs are prioritized according to functional clues regarding gene ontology, expression and function. Reverse genetics and quantitative complementation confirm the candidacy of YS1, which encodes a pentatrico-peptide-repeat (PPR) protein involved in RNA editing of plastid-encoded genes (anterograde signalling). Gene expression analysis and allele sequence comparisons reveal polymorphisms in a light-responsive element in the YS1 promoter that affect its expression, and that of its downstream targets, resulting in the variation in photosynthetic acclimation., Natural genetic variation of photosynthesis is an underexplored resource for plant genetic improvement. Here, the authors find allelic variations of YS1 affect Arabidopsis photosynthesis acclimation using genome-wide association study, reverse genetics, and quantitative complementation approaches.

To reveal the genetic variation, and loci involved, for a range of seed-related traits, a new F2 mapping population was developed by crossing Brassica rapa ssp. parachinensis L58 (CaiXin) with B. rapa ssp. trilocularis R-o-18 (spring oil seed), both rapid flowering and self-compatible. A linkage map was constructed using 97 AFLPs and 21 SSRs, covering a map distance of 757 cM with an average resolution of 6.4 cM, and 13 quantitative trait loci (QTL) were detected for nine traits. A strong seed colour QTL (LOD 26) co-localized with QTL for seed size (LOD 7), seed weight (LOD 4.6), seed oil content (LOD 6.6), number of siliques (LOD 3) and number of seeds per silique (LOD 3). There was only a significant positive correlation between seed colour and seed oil content in the yellow coloured classes. Seed coat colour and seed size were controlled by the maternal plant genotype. Plants with more siliques tended to have more, but smaller, seeds and higher seed oil content. Seed colour and seed oil content appeared to be controlled by two closely linked loci in repulsion phase. Thus, it may not always be advantageous to select for yellow-seededness when breeding for high seed oil content in Brassicas.

Plants are capable of inducing a range of physico-chemical and microbial modifications of the rhizosphere which can mobilize mineral nutrients or prevent toxic elements from entering the roots. Understanding how plants sense and adapt to variations in nutrient availability is essential in order to develop plant-based solutions addressing nutrient-use-efficiency and adaptation to nutrient-limited or -toxic soils. Recently two transcription factors of the bZIP family (basic-region leucine zipper) have been identified in Arabidopsis and shown to be pivotal in the adaptation response to zinc deficiency. They represent not only the first regulators of zinc homeostasis identified in plants, but also a very promising starting-point that can provide new insights into the molecular basis of how plants sense and adapt to the stress of zinc deficiency. Considering the available information thus far we propose in this review a putative model of how plants sense zinc deficiency.

Noccaea caerulescens of the Brassicaceae family has become the key model plant among the metal hyperaccumulator plants. Populations/accessions of N. caerulescens from geographic locations with different soil metal concentrations differ in their ability to hyperaccumulate and hypertolerate metals. Comparison of transcriptomes in several accessions provides candidates for detailed exploration of the mechanisms of metal accumulation and tolerance and local adaptation. This can have implications in the development of plants for phytoremediation and improved mineral nutrition. Transcriptomes from root and shoot tissues of four N. caerulescens accessions with contrasting Zn, Cd and Ni hyperaccumulation and tolerance traits were sequenced with Illumina Hiseq2000. Transcriptomes were assembled using the Trinity de novo assembler and were annotated and the protein sequences predicted. The comparison against the BUSCO plant early release dataset indicated high-quality assemblies. The predicted protein sequences have been clustered into ortholog groups with closely related species. The data serve as important reference sequences in whole transcriptome studies, in analyses of genetic differences between the accessions and other species, and for primer design., published version, peerReviewed

Strong selection on a beneficial mutation can cause a selective sweep, which fixes the mutation in the population and reduces the genetic variation in the region flanking the mutation [1-3]. These flanking regions have increased in frequency due to their physical association with the selected loci, a phenomenon called "genetic hitchhiking" [4]. Theoretically, selection could extend the hitchhiking to unlinked parts of the genome, to the point that selection on organelles affects nuclear genome diversity. Such indirect selective sweeps have never been observed in nature. Here we show that strong selection on a chloroplast gene in the wild plant species Arabidopsis thaliana has caused widespread and lasting hitchhiking of the whole nuclear genome. The selected allele spread more than 400 km along the British railway network, reshaping the genetic composition of local populations. This demonstrates that selection on organelle genomes can significantly reduce nuclear genetic diversity in natural populations. We expect that organelle-mediated genetic draft is a more common occurrence than previously realized and needs to be considered when studying genome evolution. Flood et al. discover that strong selection on a chloroplast gene has extended to the nuclear genome, which has hitched a ride along with the selected chloroplast. This is the first description of organelle-mediated genetic draft and shows that selection on organelles can directly impact nuclear genetic diversity.

Aims Zinc deficiency is a common micronutrient deficiency in plants growing in many different regions of the world and is associated with disturbances in uptake and accumulation of mineral nutrients. Despite many published data on physiological factors affecting ion accumulation in Zn deficient plants, there is very little information about the genetic factors underlying this. We aim to identify genetic loci involved in mineral accumulation and plant performance under Zn deficiency. Methods Genetic loci were identified using the genetically segregating Ler x Cvi recombinant inbred line (RIL) population grown under Zn deficient conditions. Lines were analysed for the concentrations of Zn, Fe, Mn, K, Ca, Mg, P, Cu, S and Al in shoot dry matter. The same was done for the same lines grown under Zn sufficient conditions. Results We found considerable heritable variation for most mineral concentrations. In general, there was a positive correlation between mineral concentrations. For Zn only condition-dependent QTLs were identified, while for most other mineral concentrations both condition-dependent and -independent QTLs were identified. Several QTLs co-localize, including co-localization to loci controlling shoot biomass and to mineral concentration loci found previously in this and other RIL populations. Conclusions There are different genetic loci controlling Zn accumulation under deficient and sufficient Zn supply. Only for few minerals, their accumulation is controlled by Zn-supply-specific loci.

Marker assisted plant breeding is a powerful technique for targeted crop improvement in horticulture and agriculture. It depends upon the correlation of desirable phenotypic characteristics with specific genetic markers. This can be determined by statistical models that relate the variation in the value of genetic markers to variation in phenotypic traits. It therefore depends upon the convergence of three technologies; the creation of genetically characterised (and thus marked) populations, high throughput screening procedures, and statistical procedures. While a large number of high throughput screening technologies are available, real-time screening techniques are usually based on some kind of imaging technologies, such as chlorophyll fluorescence imaging, that offers physiological data that are eminently suitable as a quantitative trait for genetic mapping.

The species within the now well-defined Arabidopsis genus provide biological materials suitable to investigate speciation and the development of reproductive isolation barriers between related species. Even within the model species A. thaliana, genetic differentiation between populations due to environmental adaptation or demographic history can lead to cases where hybrids between accessions are non-viable. Experimental evidence supports the importance of genome duplications and genetic epistatic interactions in the occurrence of reproductive isolation. Other examples of adaptation to specific environments can be found in Arabidopsis relatives where hybridization and chromosome doubling lead to new amphidiploid species. Molecular signals of speciation found in the Arabidopsis genus should provide a better understanding of speciation processes in plants from a genetic, molecular and evolutionary perspective.

Zinc is an essential micronutrient for all living organisms. When facing a shortage in zinc supply, plants adapt by enhancing the zinc uptake capacity. The molecular regulators controlling this adaptation are not known. We present the identification of two closely related members of the Arabidopsis thaliana basic-region leucine-zipper ( bZIP ) transcription factor gene family, bZIP19 and bZIP23 , that regulate the adaptation to low zinc supply. They were identified, in a yeast-one-hybrid screening, to associate to promoter regions of the zinc deficiency-induced ZIP4 gene of the Zrt- and Irt-related protein (ZIP) family of metal transporters. Although mutation of only one of the bZIP genes hardly affects plants, we show that the bzip19 bzip23 double mutant is hypersensitive to zinc deficiency. Unlike the wild type, the bzip19 bzip23 mutant is unable to induce the expression of a small set of genes that constitutes the primary response to zinc deficiency, comprising additional ZIP metal transporter genes. This set of target genes is characterized by the presence of one or more copies of a 10-bp imperfect palindrome in their promoter region, to which both bZIP proteins can bind. The bZIP19 and bZIP23 transcription factors, their target genes, and the characteristic cis zinc deficiency response elements they can bind to are conserved in higher plants. These findings are a significant step forward to unravel the molecular mechanism of zinc homeostasis in plants, allowing the improvement of zinc bio-fortification to alleviate human nutrition problems and phytoremediation strategies to clean contaminated soils.

This paper investigates differences in gene expression among the two Thlaspi caerulescens ecotypes La Calamine (LC) and Lellingen (LE) that have been shown to differ in metal tolerance and metal uptake. LC originates from a metalliferous soil and tolerates higher metal concentrations than LE which originates from a non-metalliferous soil. The two ecotypes were treated with different levels of zinc in solution culture, and differences in gene expression were assessed through application of a cDNA microarray consisting of 1,700 root and 2,700 shoot cDNAs. Hybridisation of root and shoot cDNA from the two ecotypes revealed a total of 257 differentially expressed genes. The regulation of selected genes was verified by quantitative reverse transcriptase polymerase chain reaction. Comparison of the expression profiles of the two ecotypes suggests that LC has a higher capacity to cope with reactive oxygen species and to avoid the formation of peroxynitrite. Furthermore, increased transcripts for the genes encoding for water channel proteins could explain the higher Zn tolerance of LC compared to LE. The higher Zn tolerance of LC was reflected by a lower expression of the genes involved in disease and defence mechanisms. The results of this study provide a valuable set of data that may help to improve our understanding of the mechanisms employed by plants to tolerate toxic concentrations of metal in the soil. © Springer-Verlag 2009.

The regulation of mineral accumulation in plants is genetically complex, with several genetic loci involved in the control of one mineral and loci affecting the accumulation of different minerals. To investigate the role of growth medium and organ type on the genetics of mineral accumulation, two existing (Ler×Kond, Ler×An-1) and one new (Ler×Eri-1) Arabidopsis thaliana Recombinant Inbred Line populations were raised on soil and hydroponics as substrates. Seeds, roots, and/or rosettes were sampled for the determination of their Ca, Fe, K, Mg, Mn, P or Zn concentrations. For seeds only, the concentration of phytate (IP6), a strong chelator of seed minerals, was determined. Correlations between minerals/IP6, populations, growth conditions, and organs were determined and mineral/IP6 concentration data were used to identify quantitative trait loci (QTLs) for these traits. A striking difference was found between QTLs identified for soil-grown versus hydroponics-grown populations and between QTLs identified for different plant organs. Three common QTLs were identified for several populations, growth conditions, and organs, one of which corresponded to the ERECTA locus, variation of which has a strong effect on plant morphology.

Cadmium (Cd) is a widespread, naturally occurring element present in soil, rock, water, plants and animals. Cd is a non-essential element for plants and is toxic at higher concentrations. Transcript profiles of roots of Arabidopsis thaliana (Arabidopsis) and Thlaspi caerulescens plants exposed to Cd and zinc (Zn) are examined, with the main aim to determine the differences in gene expression between the Cd-tolerant Zn-hyperaccumulator T. caerulescens and the Cd-sensitive non-accumulator Arabidopsis. This comparative transcriptional analysis emphasized the role of genes involved in lignin, glutathione and sulphate metabolism. Furthermore the transcription factors MYB72 and bHLH100 were studied for their involvement in metal homeostasis, as they showed an altered expression after exposure to Cd. The Arabidopsis myb72 knockout mutant was more sensitive to excess Zn or iron (Fe) deficiency than wild type, while Arabidopsis transformants overexpressing bHLH100 showed increased tolerance to high Zn and nickel (Ni) compared to wild-type plants, confirming their role in metal homeostasis in Arabidopsis. © 2008 The Authors.