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Maize is a globally valuable commodity and one of the most extensively studied genetic model organisms. However, we know surprisingly little about the extent and potential utility of the genetic variation found in wild relatives of maize. Here, we characterize a high-density genomic variation map from 744 genomes encompassing maize and all wild taxa of the genus Zea, identifying over 70 million single-nucleotide polymorphisms. The variation map reveals evidence of selection within taxa displaying novel adaptations. We focus on adaptive alleles in highland teosinte and temperate maize, highlighting the key role of flowering-time-related pathways in their adaptation. To show the utility of variants in these data, we generate mutant alleles for two flowering-time candidate genes. This work provides an extensive sampling of the genetic diversity of Zea, resolving questions on evolution and identifying adaptive variants for direct use in modern breeding.

Soil mechanical impedance precludes root penetration, confining root system development to shallow soil horizons where mobile nutrients are scarce. Using a two-phase-agar system, we characterized Arabidopsis responses to low and high mechanical impedance at three root penetration stages. We found that seedlings whose roots fail to penetrate agar barriers show a significant reduction in leaf area, root length, and elongation zone and an increment in root diameter, while those capable of penetrating show only minor morphological effects. Analyses using different auxin-responsive reporter lines, exogenous auxins, and inhibitor treatments suggest that auxin responsiveness and PIN-mediated auxin distribution play an important role in regulating root responses to mechanical impedance. The assessment of 21 Arabidopsis accessions revealed that primary root penetrability varies widely among accessions. To search for quantitative trait loci (QTLs) associated to root system penetrability, we evaluated a recombinant inbred population derived from Landsberg erecta (Ler-0, with a high primary root penetrability) and Shahdara (Sha, with a low primary root penetrability) accessions. QTL analysis revealed a major-effect QTL localized in chromosome 3, ROOT PENETRATION INDEX 3 (q-RPI3), which accounted for 29.98% (logarithm of odds=8.82) of the total phenotypic variation. Employing an introgression line (IL-321) with a homozygous q-RPI3 region from Sha in the Ler-0 genetic background, we demonstrated that q-RPI3 plays a crucial role in root penetrability. This multiscale study reveals new insights into root plasticity during the penetration process in hard agar layers, natural variation, and genetic architecture behind primary root penetrability in Arabidopsis.

Native Americans domesticated maize ( Zea mays ssp. mays ) from lowland teosinte parviglumis ( Zea mays ssp. parviglumis) in the warm Mexican southwest and brought it to the highlands of Mexico and South America where it was exposed to lower temperatures that imposed strong selection on flowering time. Phospholipids are important metabolites in plant responses to low-temperature and phosphorus availability and have been suggested to influence flowering time. Here, we combined linkage mapping with genome scans to identify High PhosphatidylCholine 1 ( HPC1 ), a gene that encodes a phospholipase A1 enzyme, as a major driver of phospholipid variation in highland maize. Common garden experiments demonstrated strong genotype-by-environment interactions associated with variation at HPC1, with the highland HPC1 allele leading to higher fitness in highlands, possibly by hastening flowering. The highland maize HPC1 variant resulted in impaired function of the encoded protein due to a polymorphism in a highly conserved sequence. A meta-analysis across HPC1 orthologs indicated a strong association between the identity of the amino acid at this position and optimal growth in prokaryotes. Mutagenesis of HPC1 via genome editing validated its role in regulating phospholipid metabolism. Finally, we showed that the highland HPC1 allele entered cultivated maize by introgression from the wild highland teosinte Zea mays ssp. mexicana and has been maintained in maize breeding lines from the Northern United States, Canada, and Europe. Thus, HPC1 introgressed from teosinte mexicana underlies a large metabolic QTL that modulates phosphatidylcholine levels and has an adaptive effect at least in part via induction of early flowering time.

Native Americans domesticated maize (

Maize is a globally valuable commodity and one of the most extensively studied genetic model organisms. However, we know surprisingly little about the extent and potential utility of the genetic variation found in the wild relatives of maize. Here, we characterize a high-density genomic variation map from 744 genomes encompassing maize and all wild taxa of the genus Zea, identifying over 70 million single nucleotide polymorphisms (SNPs) and nearly 9 million Insertion/Deletion (InDel) polymorphisms. The variation map reveals evidence of selection within taxa displaying novel adaptations to traits such as waterlogging, perenniality and regrowth. We focus in detail on adaptive alleles in highland teosinte and temperate maize and highlight the key role of flowering time related pathways in highland and high latitude adaptation. To show how this data can identify useful genetic variants, we generated and characterized novel mutant alleles for two flowering time candidate genes. This work provides the most extensive sampling to date of the genetic diversity of the genus Zea, resolving questions on evolution and identifying adaptive variants for direct use in modern breeding.

Soil mechanical impedance precludes root penetration, confining root system development to shallow soil horizons where mobile nutrients are scarce. Using a two-phase-agar system, we characterized Arabidopsis thaliana responses to low and high mechanical impedance at three root penetration stages. We found that seedlings whose roots fail to penetrate agar barriers show drastic changes in shoot and root morphology, while those capable of penetrating have only minor morphological effects. The assessment of 21 Arabidopsis accessions revealed that primary root penetrability (PRP) varies widely among accessions. To search for quantitative trait loci (QTLs) associated to root system penetrability, we evaluated a recombinant inbred population (RIL) derived from Landsberg erecta (Ler-0, with a high PRP) and Shahdara (Sha, with a low PRP) accessions. QTL analysis revealed a major-effect QTL localized in chromosome 3 (q-RPI3), which accounted for 29.98% (LOD = 8.82) of the total phenotypic variation. Employing an introgression line (IL-321), with a homozygous q-RPI3 region from Sha in the Ler-0 genetic background, we demonstrated that q-RPI3 plays a crucial role in root penetrability. This multiscale study revels new insights into root plasticity during the penetration process in hard agar layers, natural variation and genetic architecture behind primary root penetrability in Arabidopsis.HighlightWe found a wide natural variation in the capacity of Arabidopsis accessions to penetrate hard agar layers. Using a Ler-0 x Sha recombinant inbred population, a major-effect QTL (q-RPI3) strongly associated with root penetrability of compact agar layers was identified.

Plant metabolites are the basis of human nutrition and have biological relevance in ecology. Farmers selected plants with favorable characteristics since prehistoric times and improved the cultivars, but without knowledge of underlying mechanisms. Understanding the genetic basis of metabolite production can facilitate the successful breeding of plants with augmented nutritional value. To identify genetic factors related to the metabolic composition in maize, we generated mass profiles of 198 recombinant inbred lines (RILs) and their parents (B73 and Mo17) using direct-injection electrospray ionization mass spectrometry (DLI-ESI MS). Mass profiling allowed the correct clustering of samples according to genotype. We quantified 71 mass features from grains and 236 mass features from leaf extracts. For the corresponding ions, we identified tissue-specific metabolic 'Quantitative Trait Loci' (mQTLs) distributed across the maize genome. These genetic regions could regulate multiple metabolite biosynthesis pathways. Our findings demonstrate that DLI-ESI MS has sufficient analytical resolution to map mQTLs. These identified genetic loci will be helpful in metabolite-focused maize breeding. Mass profiling is a powerful tool for detecting mQTLs in maize and enables the high-throughput screening of loci responsible for metabolite biosynthesis.

Generations of farmer selection have produced a unique collection of traditional maize varieties adapted to the environmental challenges of the central Mexican highlands. In addition to agronomic and cultural value, Mexican highland maize represents a good system for the study of local adaptation and acquisition of adaptive phenotypes under cultivation. In this study, we characterized a recombinant inbred line population derived from the cross of the B73 reference line and the Mexican highland maize variety Palomero Toluqueño. Evaluation over multiple years in lowland and highland field sites in Mexico identified genomic regions linked to yield components and putatively adaptive morphological traits. A region on chromosome 7 associated with ear weight showed antagonistic allelic effects in lowland and highland fields, suggesting a trade-off consistent with local adaptation. We identified several alleles of highland origin associated with characteristic highland traits, including reduced tassel branching, increased stem pigmentation and the presence of stem macrohairs. The oligogenic architecture of characteristic morphological traits supports their role in adaptation, suggesting they have arisen from consistent directional selection acting at distinct points across the genome. We discuss these results in the context of the origin of phenotypic novelty during selection, commenting on the role ofde novomutation and the acquisition of adaptive variation by gene flow from endemic wild relatives.

Populations are locally adapted when they exhibit higher fitness than foreign populations in their native habitat. Maize landrace adaptations to highland and lowland conditions are of interest to researchers and breeders. To determine the prevalence and strength of local adaptation in maize landraces, we performed a reciprocal transplant experiment across an elevational gradient in Mexico. We grew 120 landraces, grouped into four populations (Mexican Highland, Mexican Lowland, South American Highland, South American Lowland), in Mexican highland and lowland common gardens and collected phenotypes relevant to fitness, as well as reported highland-adaptive traits such as anthocyanin pigmentation and macrohair density. 67k DArTseq markers were generated from field specimens to allow comparison between phenotypic patterns and population genetic structure.We found phenotypic patterns consistent with local adaptation, though these patterns differ between the Mexican and South American populations. While population genetic structure largely recapitulates drift during post-domestication dispersal, landrace phenotypes reflect adaptations to native elevation. Quantitative traitQSTwas greater than neutralFSTfor many traits, signaling divergent directional selection between pairs of populations. All populations exhibited higher fitness metric values when grown at their native elevation, and Mexican landraces had higher fitness than South American landraces when grown in our Mexican sites. Highland populations expressed generally higher anthocyanin pigmentation than lowland populations, and more so in the highland site than in the lowland site. Macrohair density was largely non-plastic, and Mexican landraces and highland landraces were generally more pilose. Analysis ofδ13C indicated that lowland populations may have lower WUE. Each population demonstrated garden-specific correlations between highland trait expression and fitness, with stronger positive correlations in the highland site.These results give substance to the long-held presumption of local adaptation of New World maize landraces to elevation and other environmental variables across North and South America.

Populations are locally adapted when they exhibit higher fitness than foreign populations in their native habitat. Maize landrace adaptations to highland and lowland conditions are of interest to researchers and breeders. To determine the prevalence and strength of local adaptation in maize landraces, we performed a reciprocal transplant experiment across an elevational gradient in Mexico. We grew 120 landraces, grouped into four populations (Mexican Highland, Mexican Lowland, South American Highland, South American Lowland), in Mexican highland and lowland common gardens and collected phenotypes relevant to fitness and known highland-adaptive traits such as anthocyanin pigmentation and macrohair density. 67k DArTseq markers were generated from field specimens to allow comparisons between phenotypic patterns and population genetic structure. We found phenotypic patterns consistent with local adaptation, though these patterns differ between the Mexican and South American populations. Quantitative trait differentiation (

Convergent phenotypic evolution provides some of the strongest evidence for adaptation. However, the extent to which recurrent phenotypic adaptation has arisen via parallelism at the molecular level remains unresolved, as does the evolutionary origin of alleles underlying such adaptation. Here, we investigate genetic mechanisms of convergent highland adaptation in maize landrace populations and evaluate the genetic sources of recurrently selected alleles. Population branch excess statistics reveal strong evidence of parallel adaptation at the level of individual SNPs, genes and pathways in four independent highland maize populations, even though most SNPs show unique patterns of local adaptation. The majority of selected SNPs originated via migration from a single population, most likely in the Mesoamerican highlands. Polygenic adaptation analyses of quantitative traits reveal that alleles affecting flowering time are significantly associated with elevation, indicating the flowering time pathway was targeted by highland adaptation. In addition, repeatedly selected genes were significantly enriched in the flowering time pathway, indicating their significance in adapting to highland conditions. Overall, our study system represents a promising model to study convergent evolution in plants with potential applications to crop adaptation across environmental gradients.

People living in the Balsas River basin in southwest Mexico domesticated maize from the bushy grass teosinte. Nine thousand years later, in 2021, Ms. Deb Haaland — a member of the Pueblo of Laguna tribe of New Mexico — wore a dress adorned with a cornstalk when she was sworn in as the Secretary of Interior of the United States of America. This choice of garment highlights the importance of the coevolution of maize and the farmers who, through careful selection over thousands of years, domesticated maize and adapted the physiology and shoot architecture of maize to fit local environments and growth habits. Some traits such as tillering were directly selected on (arches), and others such as tassel size are the by-products (spandrels) of maize evolution. Here, we review current knowledge of the underlying cellular, developmental, physiological, and metabolic processes that were selected by farmers and breeders, which have positioned maize as a top global staple crop.

Arbuscular mycorrhizal symbiosis is an ancient interaction between plants and fungi of the phylum Glomeromycota. In exchange for photosynthetically fixed carbon, the fungus provides the plant host with greater access to soil nutrients via an extensive network of root-external hyphae. Here, to determine the impact of the symbiosis on the host ionome, the concentration of 19 elements was determined in the roots and leaves of a panel of 30 maize varieties, grown under phosphorus-limiting conditions, with or without inoculation with the fungus Funneliformis mosseae. Although the most recognized benefit of the symbiosis to the host plant is greater access to soil phosphorus, the concentration of a number of other elements responded significantly to inoculation across the panel as a whole. In addition, variety-specific effects indicated the importance of plant genotype to the response. Clusters of elements were identified that varied in a co-ordinated manner across genotypes, and that were maintained between non-inoculated and inoculated plants.

Threats to crop production due to climate change are one of the greatest challenges facing plant breeders today. While considerable adaptive variation exists in traditional landraces, natural populations of crop wild relatives, and ex situ germplasm collections, separating adaptive alleles from linked deleterious variants that impact agronomic traits is challenging and has limited the utility of these diverse germplasm resources. Modern genome editing techniques such as CRISPR offer a potential solution by targeting specific alleles for transfer to new backgrounds, but such methods require a higher degree of precision than traditional mapping approaches can achieve. Here we present a high-resolution genome-wide association analysis to identify loci exhibiting adaptive patterns in a large panel of more than 4500 traditional maize landraces representing the breadth of genetic diversity of maize in Mexico. We evaluate associations between genotype and plant performance in 13 common gardens across a range of environments, identifying hundreds of candidate genes underlying genotype by environment interaction. We further identify genetic associations with environment across Mexico and show that such loci are associated with variation in yield and flowering time in our field trials and predict performance in independent drought trials. Our results indicate that the variation necessary to adapt crops to changing climate exists in traditional landraces that have been subject to ongoing environmental adaptation and can be identified by both phenotypic and environmental association.

Plant root systems play an essential role in nutrient and water acquisition. In resource-limited soils, modification of root system architecture is an important strategy to optimize plant performance. Most terrestrial plants also form symbiotic associations with arbuscular mycorrhizal fungi to maximize nutrient uptake. In addition to direct delivery of nutrients, arbuscular mycorrhizal fungi benefit the plant host by promoting root growth. Here, we aimed to quantify the impact of arbuscular mycorrhizal symbiosis on root growth and nutrient uptake in maize. Inoculated plants showed an increase in both biomass and the total content of twenty quantified elements. In addition, image analysis showed mycorrhizal plants to have denser, more branched root systems. For most of the quantified elements, the increase in content in mycorrhizal plants was proportional to root and overall plant growth. However, the increase in boron, calcium, magnesium, phosphorus, sulfur and strontium was greater than predicted by root system size alone, indicating fungal delivery to be supplementing root uptake.

Many important crops are members of the Poaceae family, which develop root systems characterized by a high degree of root initiation from the belowground basal nodes of the shoot, termed the crown. Although this postembryonic shoot-borne root system represents the major conduit for water uptake, little is known about the effect of water availability on its development. Here we demonstrate that in the model C4 grass Setaria viridis, the crown locally senses water availability and suppresses postemergence crown root growth under a water deficit. This response was observed in field and growth room environments and in all grass species tested. Luminescence-based imaging of root systems grown in soil-like media revealed a shift in root growth from crown-derived to primary root-derived branches, suggesting that primary root-dominated architecture can be induced in S. viridis under certain stress conditions. Crown roots of Zea mays and Setaria italica, domesticated relatives of teosinte and S. viridis, respectively, show reduced sensitivity to water deficit, suggesting that this response might have been influenced by human selection. Enhanced water status of maize mutants lacking crown roots suggests that under a water deficit, stronger suppression of crown roots actually may benefit crop productivity.

The geometries and topologies of leaves, flowers, roots, shoots, and their arrangements have fascinated plant biologists and mathematicians alike. As such, plant morphology is inherently mathematical in that it describes plant form and architecture with geometrical and topological techniques. Gaining an understanding of how to modify plant morphology, through molecular biology and breeding, aided by a mathematical perspective, is critical to improving agriculture, and the monitoring of ecosystems is vital to modeling a future with fewer natural resources. In this white paper, we begin with an overview in quantifying the form of plants and mathematical models of patterning in plants. We then explore the fundamental challenges that remain unanswered concerning plant morphology, from the barriers preventing the prediction of phenotype from genotype to modeling the movement of leaves in air streams. We end with a discussion concerning the education of plant morphology synthesizing biological and mathematical approaches and ways to facilitate research advances through outreach, cross-disciplinary training, and open science. Unleashing the potential of geometric and topological approaches in the plant sciences promises to transform our understanding of both plants and mathematics. National Science Foundation through NSF [DBI-1300426] University of Tennessee, Knoxville, Knoxville, TN, United States This work was assisted through participation in the Morphological Plant Modeling: Unleashing geometric and topological potential within the plant sciences Investigative Workshop at the National Institute for Mathematical and Biological Synthesis, sponsored by the National Science Foundation through NSF Award #DBI-1300426, with additional support from The University of Tennessee, Knoxville, Knoxville, TN, United States.

Arbuscular mycorrhizal symbiosis is an ancient interaction between plants and fungi of the phylum Glomeromycota. In exchange for photosynthetically fixed carbon, the fungus provides the plant host with greater access to soil nutrients via an extensive network of root-external hyphae. Here, to determine the impact of the symbiosis on the host ionome, the concentration of nineteen elements was determined in the roots and leaves of a panel of thirty maize varieties, grown under phosphorus limiting conditions, with, or without, inoculation with the fungus Funneliformis mosseae. Although the most recognized benefit of the symbiosis to the host plant is greater access to soil phosphorus, the concentration of a number of other elements responded significantly to inoculation across the panel as a whole. In addition, variety-specific effects indicated the importance of plant genotype to the response. Clusters of elements were identified that varied in a coordinated manner across genotypes, and that were maintained between non-inoculated and inoculated plants.AbbreviationsNCnon-colonizedMmycorrhizalSDWshoot dry weightICP-MSinductively coupled plasma mass spectrometryPCprincipal component

Low phosphate (Pi) availability constrains plant development and seed production in both natural and agricultural ecosystems. When Pi is scarce, modifications of root system architecture (RSA) enhance the soil exploration ability of the plant and lead to an increase in Pi uptake. In Arabidopsis, an iron-dependent mechanism reprograms primary root growth in response to low Pi availability. This program is activated upon contact of the root tip with low-Pi media and induces premature cell differentiation and the arrest of mitotic activity in the root apical meristem, resulting in a short-root phenotype. However, the mechanisms that regulate the primary root response to Pi-limiting conditions remain largely unknown. Here we report on the isolation and characterization of two low-Pi insensitive mutants (lpi5 and lpi6), which have a long-root phenotype when grown in low-Pi media. Cellular, genomic, and transcriptomic analysis of low-Pi insensitive mutants revealed that the genes previously shown to underlie Arabidopsis Al tolerance via root malate exudation, known as SENSITIVE TO PROTON RHIZOTOXICITY (STOP1) and ALUMINUM ACTIVATED MALATE TRANSPORTER 1 (ALMT1), represent a critical checkpoint in the root developmental response to Pi starvation in Arabidopsis thaliana Our results also show that exogenous malate can rescue the long-root phenotype of lpi5 and lpi6 Malate exudation is required for the accumulation of Fe in the apoplast of meristematic cells, triggering the differentiation of meristematic cells in response to Pi deprivation.

Plant morphology is inherently mathematical in that morphology describes plant form and architecture with geometrical and topological descriptors. The geometries and topologies of leaves, flowers, roots, shoots and their spatial arrangements have fascinated plant biologists and mathematicians alike. Beyond providing aesthetic inspiration, quantifying plant morphology has become pressing in an era of climate change and a growing human population. Modifying plant morphology, through molecular biology and breeding, aided by a mathematical perspective, is critical to improving agriculture, and the monitoring of ecosystems with fewer natural resources. In this white paper, we begin with an overview of the mathematical models applied to quantify patterning in plants. We then explore fundamental challenges that remain unanswered concerning plant morphology, from the barriers preventing the prediction of phenotype from genotype to modeling the movement of leafs in air streams. We end with a discussion concerning the incorporation of plant morphology into educational programs. This strategy focuses on synthesizing biological and mathematical approaches and ways to facilitate research advances through outreach, cross-disciplinary training, and open science. This white paper arose from bringing mathematicians and biologists together at the National Institute for Mathematical and Biological Synthesis (NIMBioS) workshop titled “Morphological Plant Modeling: Unleashing Geometric and Topological Potential within the Plant Sciences” held at the University of Tennessee, Knoxville in September, 2015. Never has the need to quantify plant morphology been more imperative. Unleashing the potential of geometric and topological approaches in the plant sciences promises to transform our understanding of both plants and mathematics.

25 Pag., 2 Tabl., 5 Fig. The definitive version is available at: http://www.elsevier.de/jplph, Iron deficiency induces several mechanisms in response to iron shortage in plants. Metabolic changes occur to sustain the increased iron uptake capacity of Fe-deficient plants. We evaluated the metabolic changes of three Prunus rootstocks submitted to iron chlorosis and their different response for tolerance using measurements of metabolites and enzymatic activities. The more tolerant rootstocks Adesoto (Prunus insititia) and GF 677 (Prunus amygdalus × Prunus persica), and the more sensitive Barrier (P. persica × Prunus davidiana) were grown hydroponically in iron sufficient and deficient conditions during two weeks. Sugar, organic and amino acid concentrations of root tips were determined after two weeks of iron shortage by proton nuclear magnetic resonance spectroscopy of extracts. Complementary analyses of organic acids were performed by liquid chromatography coupled to mass spectrometry. The major soluble sugars found were glucose and sucrose. The major organic acids were malic and citric acids, and the major amino acid was asparagine. Iron deficiency increased root sucrose, total organic and amino acid concentrations and phosphoenolpyruvate carboxylase activity. After two weeks of iron deficiency, the malic, citric and succinic acid concentrations increased in the three rootstocks, although no significant differences were found among genotypes with different tolerance to iron chlorosis. The tolerant rootstock Adesoto showed higher total organic and amino acid concentrations. In contrast, the susceptible rootstock Barrier showed lower total amino acid concentration and phosphoenolpyruvate carboxylase activity values. These results suggest that the induction of this enzyme activity under iron deficiency, as previously shown in herbaceous plants, indicates the tolerance level of rootstocks to iron chlorosis. The analysis of other metabolic parameters, such as organic and amino acid concentrations, gives complementary information for selection of genotypes tolerant to iron chlorosis., This work was partly funded by the Spanish-French bilateral cooperation programs (Picasso Program-HF2003-273; Aquitaine- Aragón Aq23 and Aq25) and the Spanish MICINN (Ministerio de Ciencia e Innovación, grants AGL-2005-05533, AGL-2008-00283 and AGL2009-09018). Sergio Jiménez was supported by an I3P fellowship from CSIC/FSE (Consejo Superior de Investigaciones Científicas/ Fondo Social Europeo) and a travel fellowship from DGA/CAI (CA 5/03).

The plant root system traverses one of the most complex environments on earth. Understanding how roots support plant life on land requires knowing how soil properties affect the availability of nutrients and water and how roots manipulate the soil environment to optimize acquisition of these resources. Imaging of roots in soil allows the integrated analysis and modeling of environmental interactions occurring at micro- to macroscales. Advances in phenotyping of root systems is driving innovation in cross-platform-compatible methods for data analysis. Root systems acclimate to the environment through architectural changes that act at the root-type level as well as through tissue-specific changes that affect the metabolic needs of the root and the efficiency of nutrient uptake. A molecular understanding of the signaling mechanisms that guide local and systemic signaling is providing insight into the regulatory logic of environmental responses and has identified points where crosstalk between pathways occurs.

Root systems develop different root types that individually sense cues from their local environment and integrate this information with systemic signals. This complex multi-dimensional amalgam of inputs enables continuous adjustment of root growth rates, direction, and metabolic activity that define a dynamic physical network. Current methods for analyzing root biology balance physiological relevance with imaging capability. To bridge this divide, we developed an integrated-imaging system called Growth and Luminescence Observatory for Roots (GLO-Roots) that uses luminescence-based reporters to enable studies of root architecture and gene expression patterns in soil-grown, light-shielded roots. We have developed image analysis algorithms that allow the spatial integration of soil properties, gene expression, and root system architecture traits. We propose GLO-Roots as a system that has great utility in presenting environmental stimuli to roots in ways that evoke natural adaptive responses and in providing tools for studying the multi-dimensional nature of such processes. DOI: http://dx.doi.org/10.7554/eLife.07597.001, eLife digest Most plants absorb water and nutrients from the soil via structures called roots. The shape, size, and structure of a plant's root system can change over its lifetime as the plant responds to changes in their local environment. For example, if water is scarce, a plant may develop a very deep root system that is more efficient at capturing water. Understanding how root systems respond to environmental cues may help us to identify the genes and processes involved. In this study, Rellán-Álvarez et al. report a new live-imaging platform for analyzing root architecture and its regulation. This platform is called Growth and Luminescence Observatory for Roots (or GLO-Roots for short) and uses ‘luminescent’ markers that allow growing roots to be visualized when plants are grown in thin, soil-filled, transparent pots. GLO-Roots can track the growth of the plant roots as well as the activity of genes that respond to environmental stress. Rellán-Álvarez et al. developed a software tool called GLO-RIA (GLO-Roots Image Analysis) to analyze the resulting images. GLO-RIA performs several different types of image analysis, including one that detects the position, length, and direction of roots, as well as their shape and depth. Rellán-Álvarez et al. tested the GLO-Roots techniques in various ways, for example, by analyzing the effects that different conditions have on the growth of the roots of the model plant known as Arabidopsis thaliana. Depriving the plants of a nutrient called phosphorous caused the roots to grow more horizontally than when phosphorus is plentiful, presumably to allow the plants to expand their search for phosphate in the upper layers of the soil, where this nutrient is usually more abundant. On the other hand, a shortage of water caused the roots to grow more vertically to access water stored deeper in the soil. GLO-Roots can also be used to measure the water content of soil at different depths and how this influences the architecture of the root. Further experiments on tomato plants and a grass species called Brachypodium distachyon revealed the different architectures of their root systems. Rellán-Álvarez et al. propose that this system could be used to study the roots of other plant species in a variety of environmental conditions. This will provide a more detailed understanding of the ways that different plants adapt in response to changes in their environment. DOI: http://dx.doi.org/10.7554/eLife.07597.002

11 pages, 5 figures, 4 tables.-- PMID: 16828049 [PubMed].-- Printed version published Sep 15, 2006., A simple, highly selective, sensitive, and reproducible liquid chromatography–electrospray ionization/mass spectrometry (time of flight) method has been developed for the direct and simultaneous determination of glutathione and related compounds such as homoglutathione in different plant tissues. These compounds are low-molecular mass antioxidants involved in cellular redox homeostasis in plants, and efforts are being made to develop methods to determine the concentrations of oxidized and reduced forms of these compounds and their ratio. Many of the methodologies developed so far, however, are time-consuming and complex; therefore, analytes can decompose and their redox status can change during the analysis process. The method we have developed allows the simultaneous determination of reduced forms (glutathione [GSH] and homoglutathione [hGSH]) and oxidized forms (glutathione disulfide [GSSG]) of these compounds and is also suitable for the determination of ascorbic acid (ASA) and S-nitrosoglutathione (GSNO). Quantification was done using isotopically labeled GSH and ASA as internal standards. All compounds were base peak resolved in less than 6 min, and limits of detection were 60 pmol for GSH, 30 pmol for hGSH, 20 pmol for GSSG, 100 pmol for ASA, and 30 pmol for GSNO. The intraday repeatability values were approximately 0.4 and 7% for retention time and peak area, respectively, whereas the interday repeatability values were approximately 0.6 and 9% for retention time and peak area, respectively. Analyte recoveries found were between 92 and 105%. The method was used to determine the concentrations of GSH, GSSG, hGSH, and ASA in extracts from several plant tissues., This work was funded by projects from the Spanish Ministry of Education and Science (MEC) (REN2001-113-C01-02 and REN2002-04229-C02-01 to L. E. Hernández and AGL2004-00194 [cofinanced with FEDER] to J. Abadía). R. Rellán-Álvarez was supported by the Universidad Autónoma de Madrid through a grant of the Ph.D. Student Exchange Program and an FPI fellowship of the Spanish MEC. A. Álvarez-Fernández was supported by a “Ramón y Cajal” research contract from the Spanish MEC. Acquisition of the HPLC/MS(TOF) apparatus was cofinanced with FEDER.

Alfalfa (Medicago sativa) plantlets were exposed to Cd or Hg to study the kinetics of diverse stress indexes. In the so-called beaker-size hydroponic system, plantlets were grown in 30 microM of Cd or Hg for 7 d. Oxidative stress took place and increased over time, a linear response being observed with Cd but not with Hg. To improve the sensitivity of the stress assays used, a micro-assay system, in which seedlings were exposed for 24 h, was developed. Phytotoxicity of metals, quantified as growth inhibition, was observed well before there was any change in the non-protein thiol tissue concentration. When measured with conventional techniques, oxidative stress indexes did not show significant variation. To trace early and small plant responses to Cd and Hg, a microscopic analysis with novel fluorescent dyes, which had not yet been exploited to any significant extent for use in plants, was conducted. These fluorescent probes, which allowed minute cellular responses to 0, 3, 10, and 30 microM of both metals to be visualized in the roots of the alfalfa seedlings, were: (i) 2',7'-dichlorofluorescin diacetate that labels peroxides; (ii) monochlorobimane that stains reduced glutathione/homoglutathione (GSH/hGSH); and (iii) propidium iodide that marks nuclei of dead cells. Oxidative stress and cell death increased after exposure for 6-24 h to Cd and Hg, but labelling of GSH/hGSH decreased acutely. This diminution might be the result of direct interaction of GSH/hGSH with both Cd and Hg, as inferred from an in vitro conjugation assay. Therefore, both Cd and Hg not only compromised severely the cellular redox homeostasis, but also caused cell necrosis. In plants treated with 1 mM L-buthionine sulphoximine, a potent inhibitor of GSH/hGSH synthesis, only the oxidative stress symptoms appeared, indicating that the depletion of the GSH/hGSH pool was not sufficient to promote cell death, and that other phytotoxic mechanisms might be involved.

24 Pág., 2 Fig., 9 Tabl. The definitive version is available at: http://jxb.oxfordjournals.org/content/64/10/2665, This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited, Iron homeostasis is an important process for flower development and plant fertility. The role of plastids in these processes has been shown to be essential. To document the relationships between plastid iron homeostasis and flower biology further, a global study (transcriptome, proteome, metabolome, and hormone analysis) was performed of Arabidopsis flowers from wild-type and triple atfer1-3-4 ferritin mutant plants grown under iron-sufficient or excess conditions. Some major modifications in specific functional categories were consistently observed at these three omic levels, although no significant overlaps of specific transcripts and proteins were detected. These modifications concerned redox reactions and oxidative stress, as well as amino acid and protein catabolism, this latter point being exemplified by an almost 10-fold increase in urea concentration of atfer1-3-4 flowers from plants grown under iron excess conditions. The mutant background caused alterations in Fehaem redox proteins located in membranes and in hormone-responsive proteins. Specific effects of excess Fe in the mutant included further changes in these categories, supporting the idea that the mutant is facing a more intense Fe/redox stress than the wild type. The mutation and/or excess Fe had a strong impact at the membrane level, as denoted by the changes in the transporter and lipid metabolism categories. In spite of the large number of genes and proteins responsive to hormones found to be regulated in this study, changes in the hormonal balance were restricted to cytokinins, especially in the mutant plants grown under Fe excess conditions. © The Author 2013., This work was supported in the framework of the European Transnational Cooperation (Germany, France, Spain) within the PLANT-KBBE Initiative funded by (i) the Bundesministerium für Bildung und Forschung (BMBF, framework of the GABI initiative, FKZ:0315458A to KP), (ii) the Spanish Ministry of Economy and Competitivity (MINECO EUI2008-03618 to JA.) and (iii) the Agence Nationale de la Recherche (ANR-08-KBBE-009-01 to J-FB). The study was also supported by the MINECO grant AGL2010-16515 to JA.

18 Pags, 5 Tabls., 7 Figs., 1 Appendix with 1 Tabl. The .pdf Document is Protected by copyright and was first published by Frontiers., The metabolite profile changes induced by Fe deficiency in leaves and xylem sap of several Strategy I plant species have been characterized. We have confirmed that Fe deficiency causes consistent changes both in the xylem sap and leaf metabolite profiles. The main changes in the xylem sap metabolite profile in response to Fe deficiency include consistent decreases in amino acids, N-related metabolites and carbohydrates, and increases in TCA cycle metabolites. In tomato, Fe resupply causes a transitory flush of xylem sap carboxylates, but within 1 day the metabolite profile of the xylem sap from Fe-deficient plants becomes similar to that of Fe-sufficient controls. The main changes in the metabolite profile of leaf extracts in response to Fe deficiency include consistent increases in amino acids and N-related metabolites, carbohydrates and TCA cycle metabolites. In leaves, selected pairs of amino acids and TCA cycle metabolites show high correlations, with the sign depending of the Fe status. These data suggest that in low photosynthesis, C-starved Fe-deficient plants anaplerotic reactions involving amino acids can be crucial for short-term survival., This study was supported by the Spanish Ministry of Science and Innovation (MICINN; projects AGL2009-09018 and AGL2010-16515, co-financed with FEDER), the trilateral Project Hot Iron (ERA-NET Plant Genome Research KKBE; MICINN EUI2008-03618), the Spanish National Research Council (PA1000990, International Promotion Programme 2007), and the Aragón Government (group A03). Rubén Rellán-Álvarez and Hamdi El-Jendoubi were supported by MICINN-FPI grants.

7 Pag., 5 Tabl., 2 Fig., A liquid chromatography–electrospray ionization time-of-flight mass spectrometry method has been developed for the direct and simultaneous determination of 10 low molecular mass organic acids in different plant tissue extracts. The method does not include a derivatization step. Quantification was accomplished using 13C-labeled malic and succinic acids as internal standards. Good limits of detection (0.05–1.27 pmol) were obtained for malic, 2-oxoglutaric, succinic, quinic, shikimic, cis-aconitic, and citric acids, whereas for oxalic, ascorbic, and fumaric acids limits of detection were 255, 25, and 11 pmol, respectively. Repeatability values for the retention time and peak area were, This study was supported by the Spanish Ministry of Science and Innovation (MICINN; Projects AGL2007-61948 and AGL2009-09018, cofinanced with FEDER), the European Commission (Thematic Priority 5—Food Quality and Safety, sixth Framework RTD Programme, Contract FP6-FOOD-CT-2006-016279), the trilateral Project Hot Iron (ERA-NET Plant Genome Research KKBE; MICINN Project EUI2008-03618), and the Aragn Government (group A03). R.R.-A. and S.L.-G. were supported by an FPI-MCINN fellowship and a CSIC Grant (I3P-2005) from the I3P programme, cofinanced by the European Social Fund, respectively. Acquisition of the HPLC–TOFMS apparatus was cofinanced with FEDER.

4 Pag., 2 Tabl., 1 Fig. The definitive version is available at: http://www.sciencedirect.com/science/journal/01761617, The effects of Zn excess on carboxylate metabolism were investigated in sugar beet (Beta vulgaris L.) plants grown hydroponically in a growth chamber. Root extracts of plants grown with 50 or 100 M Zn in the nutrient solution showed increases in several enzymatic activities related to organic acid metabolism, including citrate synthase and phosphoenolpyruvate carboxylase, when compared to activities in control root extracts. Root citric and malic acid concentrations increased in plants grown with 100 M Zn, but not in plants grown with 50 M Zn. In the xylem sap, plants grown with 50 and 100 M Zn showed increases in the concentrations of citrate and malate compared to the controls. Leaves of plants grown with 50 or 100 MZn showed increases in the concentrations of citric and malic acid and in the activities of citrate synthase and fumarase. Leaf isocitrate dehydrogenase increased only in plants grown with 50 M Zn when compared to the controls. In plants grown with 300 M Zn, the only enzyme showing activity increases in root extracts was citrate synthase, whereas the activities of other enzymes decreased compared to the controls, and root citrate concentrations increased. In the 300 MZn-grown plants, the xylem concentrations of citric and malic acids were higher than those of controls, whereas in leaf extracts the activity of fumarase increased markedly, and the leaf citric acid concentration was higher than in the controls. Based on our data, a metabolic model of the carboxylate metabolism in sugar beet plants grown under Zn excess is proposed., This work was supported by the Spanish Ministry of Science and Innovation (grant AGL2007-61948, co-financed with FEDER) and the Aragón Government (group A03). The HPLC–TOFMS apparatus was co-financed with FEDER. R.S. and R.R-A were supported by I3P-CSIC and FPI-MCINN pre-doctoral fellowships, respectively.

12 Pag., 8 Fig. The definitive version is available at: www.sciencedirect.com/science/journal/09819428, Iron (Fe) deficiency-induced chlorosis is a major nutritional disorder in crops growing in calcareous soils. Iron deficiency in fruit tree crops causes chlorosis, decreases in vegetative growth and marked fruit yield and quality losses. Therefore, Fe fertilizers, either applied to the soil or delivered to the foliage, are used every year to control Fe deficiency in these crops. On the other hand, a substantial body of knowledge is available on the fundamentals of Fe uptake, long and short distance Fe transport and subcellular Fe allocation in plants. Most of this basic knowledge, however, applies only to Fe deficiency, with studies involving Fe fertilization (i.e., with Fe-deficient plants resupplied with Fe) being still scarce. This paper reviews recent developments in Fe-fertilizer research and the state-of-the-art of the knowledge on Fe acquisition, transport and utilization in plants. Also, the effects of Fe-fertilization on the plant responses to Fe deficiency are reviewed. Agronomical Fe-fertilization practices should benefit from the basic knowledge on plant Fe homeostasis already available; this should be considered as a long-term goal that can optimize fertilizer inputs, reduce grower’s costs and minimize the environmental impact of fertilization., This studywas supported by the Spanish Ministry of Science and Innovation (MICINN; projects AGL2007-61948 and AGL2009- 09018, co-financed with FEDER), the European Commission (Isafruit Thematic Priority 5eFood Quality and Safety, 6th Framework RTD Programme, Contract no. FP6-FOODeCT-2006-016279), the trilateral Project Hot Iron (ERA-NET Plant Genome Research KKBE; MICINN EUI2008-03618), and the Aragón Government (group A03). HE-J and SV were supported by an FPI-MICINN fellowship and an I3P-CSIC postdoctoral contract, respectively

13 Pags., 1 Tabl., 4 Figs., Tomato is an important crop from nutritional and economical points of view, and it is grown in greenhouses, where special substrates and the use of recycled water imply an increased risk of Cd accumulation. We investigated tomato root responses to low (10 µM) and high (100 µM) Cd concentrations at the root proteome level. Root extract proteome maps were obtained by 2-DE, and an average of 121, 145 and 93 spots were detected in the 0, 10 and 100 µM Cd treatments, respectively. The low Cd treatment (10 µM) resulted in significant and higher than 2-fold changes in the relative amounts of 36 polypeptides, with 27 of them identified by mass spectrometry, whereas the 100 µM Cd treatment resulted in changes in the relative amounts of 41 polypeptides, with 33 of them being identified. The 2-DE based proteomic approach allowed assessing the main metabolic pathways affected by Cd toxicity. Our results suggests that the 10 µM Cd treatment elicits proteomic responses similar to those observed in Fe deficiency, including activation of the glycolytic pathway, TCA cycle and respiration, whereas the 100 µM Cd treatment responses are more likely due to true Cd toxicity, with a general shutdown of carbon metabolism and increases in stress related and detoxification proteins., JRC and RRA were supported by I3P and FPI predoctoral fellowships from the CSIC and MCINN (Spanish Ministry of Science and Innovation), respectively. This work was supported by the MICINN (project AGL2007-61948, cofinanced with FEDER) and the Aragón Government (group A03).

9 Pags., Tabls., Figs., Organic wastes such as sewage sludge have been successfully used to increase crop productivity of horticultural soils. Nevertheless, considerations of the impact of sludges on vegetable and fruit quality have received little attention. Therefore, the objective of the present work was to investigate the impact of two sanitized sewage sludges, autothermal thermophilic aerobic digestion (ATAD) and compost sludge, on the growth, yield, and fruit quality of pepper plants (Capsicum annuum L. cv. Piquillo) grown in the greenhouse. Two doses of ATAD (15 and 30% v/v) and three of composted sludge (15, 30, and 45%) were applied to a peat-based potting mix. Unamended substrate was included as control. ATAD and composted sludge increased leaf, shoot, and root dry matter, as well as fruit yield, mainly due to a higher number of fruits per plant. There was no effect of sludge on fruit size (dry matter per fruit and diameter). The concentrations of Zn and Cu in fruit increased with the addition of sewage sludges. Nevertheless, the levels of these elements remained below toxic thresholds. Pepper fruits from sludge-amended plants maintained low concentrations of capsaicin and dihydrocapsaicin, thus indicating low pungency level, in accordance with the regulations prescribed by the Control Board of “Lodosa Piquillo peppers” Origin Denomination. The application of sludges did not modify the concentration of vitamin C (ASC) in fruit, whereas the highest doses of composted sludge tended to increase the content of reduced (GSH) and oxidized (GSSG) glutathione, without change in the GSH/GSSG ratio. There were no effects of sludge on the transcript levels of enzymes involved in the synthesis of vitamin C, l-galactono-1,4-lactone dehydrogenase (GLDH) or in the ascorbate−glutathione cycle, ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), and glutathione reductase (GR). Results suggest that the synthesis and degradation of ASC and GSH were compensated for in most of the treatments assayed. The application of sanitized sludges to pepper plants can improve pepper yield without loss of food nutritional quality, in terms of fruit size and vitamin C, glutathione, and capsaicinoid contents.

12 páginas, 5 figuras, 2 tablas., The identification of Fe transport forms in plant xylem sap is crucial to the understanding of long-distance Fe transport processes in plants. Previous studies have proposed that Fe may be transported as an Fe–citrate complex in plant xylem sap, but such a complex has never been detected. In this study we report the first direct and unequivocal identification of a natural Fe complex in plant xylem sap. A tri-Fe(III), tri-citrate complex (Fe3Cit3) was found in the xylem sap of Fe-deficient tomato (Solanum lycopersicum Mill. cv. ‘Tres Cantos’) resupplied with Fe, by using an integrated mass spectrometry approach based on exact molecular mass, isotopic signature and Fe determination and retention time. This complex has been modeled as having an oxo-bridged tri-Fe core. A second complex, a di-Fe(III), di-citrate complex was also detected in Fe–citrate standards along with Fe3Cit3, with the allocation of Fe between the two complexes depending on the Fe to citrate ratio. These results provide evidence for Fe–citrate complex xylem transport in plants. The consequences for the role of Fe to citrate ratio in long-distance transport of Fe in xylem are also discussed., This work was supported by the Spanish Ministry of Science and Innovation (grant numbers AGL2006-1416, AGL2007-61948 and CTQ2006-05722, co-financed with FEDER); the European Commission (Thematic Priority 5—Food Quality and Safety, 6th Framework RTD Programme, grant number FP6-FOOD-CT-2006-016279) and the Aragón Government (groups A03 and E39). Acquisition of the HPLC–TOFMS apparatus was cofinanced with FEDER. R.R.-A., J.G.-M.-S., I.O. and J.A.R.-C. were supported by FPI-MICINN, FICYT-MICINN, CONAID-DGA and FPU-MICINN grants, respectively.

7 Pags.- 6 Tabl.- 1 Fig., The refining process in the petrochemical industry generates oil refinery sludges, a potentially contaminating waste product, with a high content of hydrocarbons and heavy metals. Faster degradation of hydrocarbons has been reported in vegetated soils than in non-vegetated soils, but the impact of these contaminants on the plants physiology and on their antioxidant system is not well known. In this study, the effect of the addition of petroleum sludge to soil on the physiological parameters, nutrient contents, and oxidative and antioxidant status in alfalfa was investigated. An inhibition of alfalfa growth and an induction of oxidative stress, as indicated by an increase in protein oxidation, were found. Also, the superoxide dismutase isoenzymes, peroxidase, and those enzymes involved in the ascorbate–glutathione cycle showed significant activity increases, parallel to an enhancement of total homoglutathione, allowing plants being tolerant to this situation. This information is necessary to establish successful and sustainable plant-based remediation strategies., The research was financially supported by SÉNECA Foundation (project 00570/PI/04 and M.C. Martí fellowship, co-financed with SREF and FSE), by the Spanish Ministry of Science and Innovation (project AGL2007-61948, co-financed with FEDER), and the Aragón Government (group A03).

Nicotianamine (NA) is considered as a key element in plant metal homeostasis. This non-proteinogenic amino acid has an optimal structure for chelation of metal ions, with six functional groups that allow octahedral coordination. The ability to chelate metals by NA is largely dependent on the pK of the resulting complex and the pH of the solution, with most metals being chelated at neutral or basic pH values. In silico calculations using pKa and pK values have predicted the occurrence of metal-NA complexes in plant fluids, but the use of soft ionization techniques (e.g. electrospray), together with high-resolution mass spectrometers (e.g. time-of-flight mass detector), can offer direct and metal-specific information on the speciation of NA in solution. We have used direct infusion electrospray ionization mass spectrometry (time-of-flight) ESI-MS(TOF) to study the complexation of Mn, Fe(II), Fe(III), Ni, Cu by NA. The pH dependence of the metal-NA complexes in ESI-MS was compared to that predicted in silico. Possible exchange reactions that may occur between Fe-NA and other metal micronutrients as Zn and Cu, as well as between Fe-NA and citrate, another possible Fe ligand candidate in plants, were studied at pH 5.5 and 7.5, values typical of the plant xylem and phloem saps. Metal-NA complexes were generally observed in the ESI-MS experiments at a pH value approximately 1-2 units lower than that predicted in silico, and this difference could be only partially explained by the estimated error, approximately 0.3 pH units, associated with measuring pH in organic solvent-containing solutions. Iron-NA complexes are less likely to participate in ligand- and metal-exchange reactions at pH 7.5 than at pH 5.5. Results support that NA may be the ligand chelating Fe at pH values usually found in phloem sap, whereas in the xylem sap NA is not likely to be involved in Fe transport, conversely to what occurs with other metals such as Cu and Ni. Some considerations that need to be addressed when studying metal complexes in plant compartments by ESI-MS are also discussed. Copyright © 2008 John Wiley & Sons, Ltd., This work was supported by the Spanish Ministry of Science and Education (Grant Nos. AGL2006-1416 and AGL2007-61948, co-financed with FEDER), the European Commission (EU 6th Framework Integrated Project ISA-FRUIT), and the Aragón Government (group A03).

The American Society of Plant Biologists (ASPB) “supports the continued responsible use of genetic engineering… as an effective tool for advancing food security and reducing the negative environmental impacts of agriculture” ([ 1 ][1]). A recent petition advocating the ASPB position collected

• Here, the kinetics of oxidative stress responses of alfalfa (Medicago sativa) seedlings to cadmium (Cd) and mercury (Hg) (0, 3, 10 and 30 μm) exposure, expanding from a few minutes to 24 h, were studied. • Intracellular oxidative stress was analysed using 2′,7′- dichlorofluorescin diacetate and extracellular hydrogen peroxide (H 2O2) production was studied with Amplex Red. Growth inhibition, concentrations of ascorbate, glutathione (GSH), homoglutathione (hGSH), Cd and Hg, ascorbate peroxidase (APX) activity, and expression of genes related to GSH metabolism were also determined. • Both Cd and Hg increased cellular reactive oxygen species (ROS) production and extracellular H 2O2 formation, but in different ways. The increase was mild and slow with Cd, but more rapid and transient with Hg. Hg treatments also caused a higher cell death rate, significant oxidation of hGSH, as well as increased APX activity and transient overexpression of glutathione reductase 2, glutamylcysteinyl synthetase, and homoglutathione synthetase genes. However, Cd caused minor alterations. Hg accumulation was one order of magnitude higher than Cd accumulation. • The different kinetics of early physiological responses in vivo to Cd and Hg might be relevant to the characterization of their mechanisms of toxicity. Thus, high accumulation of Hg might explain the metabolism poisoning observed in Hg-treated seedlings., The authors gratefully acknowledge the financial support of the Fundación Ramón Areces, which made possible this work, specially the PhD grant received by COV. This work was also supported by the Spanish Ministry of Education and Science through the projects REUSA (CTM2005-04809/TECNO) and AGL2004-00191. RRA was supported by a FPI PhD grant from the Spanish Ministry of Education and Science.

A hydroponic experiment was carried out to characterize the oxidative stress responses of maize seedlings (Zea mays L. cv. Dekalb DK604) to cadmium (Cd) and mercury (Hg). Plants were grown hydroponically for 7 days in a nutrient solution supplemented with several concentrations of Cd and Hg: 0.0 (control), 6 or 30 μM. Growth was inhibited by both metals. The effect was more severe in plants exposed to Hg. Oxidative stress was caused by the exposure to the metals, as quantified by malondialdehyde and carbonyl accumulation, by-products of lipid peroxidation and protein oxidation, respectively. The activity of ascorbate peroxidase (APX) and superoxide dismutase (SOD), enzymes involved in the scavenging of reactive oxygen species, were measured upon metal treatment. We found an activation of a cytosolic APX isoform, as identified by using a specific polyclonal antiserum. However, there were negligible changes in SOD activity. Analysis of thiol-peptides revealed that at 6 μM Cd a remarkable increase in root reduced glutathione (GSH) content occurred, and little effect on the relative content of oxidised glutathione (GSSG) was observed. However, at 30 μM Cd and in plants exposed to 6 and 30 μM of Hg, GSH root content either remained stable or decreased significantly, while the proportion of GSSG increased. Moreover, only Cd was able to induce accumulation of phytochelatins at both assayed concentrations. Apparently, Hg was more toxic than Cd, as inferred from the magnitude of the changes found in the physiological parameters tested. © Springer 2006., Funding from the Spanish Ministry of Education and Science (REN2001-113-C01-02 and REN2002-04229-C02-01). R.R.A. recognises the funding from UAM, through a PhD Student Exchange Program grant.

15 páginas, 4 figuras, 2 tablas.-- et al., [Background]: Plants grown under iron deficiency show different morphological, biochemical and physiological changes. These changes include, among others, the elicitation of different strategies to improve the acquisition of Fe from the rhizosphere, the adjustment of Fe homeostasis processes and a reorganization of carbohydrate metabolism. The application of modern techniques that allow the simultaneous and untargeted analysis of multiple proteins and metabolites can provide insight into multiple processes taking place in plants under Fe deficiency. The objective of this study was to characterize the changes induced in the root tip proteome and metabolome of sugar beet plants in response to Fe deficiency and resupply. [Results]: Root tip extract proteome maps were obtained by 2-D isoelectric focusing polyacrylamide gel electrophoresis, and approximately 140 spots were detected. Iron deficiency resulted in changes in the relative amounts of 61 polypeptides, and 22 of them were identified by mass spectrometry (MS). Metabolites in root tip extracts were analyzed by gas chromatography-MS, and more than 300 metabolites were resolved. Out of 77 identified metabolites, 26 changed significantly with Fe deficiency. Iron deficiency induced increases in the relative amounts of proteins and metabolites associated to glycolysis, tri-carboxylic acid cycle and anaerobic respiration, confirming previous studies. Furthermore, a protein not present in Fe-sufficient roots, dimethyl-8-ribityllumazine (DMRL) synthase, was present in high amounts in root tips from Fe-deficient sugar beet plants and gene transcript levels were higher in Fe-deficient root tips. Also, a marked increase in the relative amounts of the raffinose family of oligosaccharides (RFOs) was observed in Fe-deficient plants, and a further increase in these compounds occurred upon short term Fe resupply. [Conclusions]: The increases in DMRL synthase and in RFO sugars were the major changes induced by Fe deficiency and resupply in root tips of sugar beet plants. Flavin synthesis could be involved in Fe uptake, whereas RFO sugars could be involved in the alleviation of oxidative stress, C trafficking or cell signalling. Our data also confirm the increase in proteins and metabolites related to carbohydrate metabolism and TCA cycle pathways., RRA and SA were supported by FPI fellowships from the Spanish Ministry of Science and Innovation (MICINN). JRC was supported by an I3P-CSIC predoctoral fellowship. Work supported by the Spanish MICINN (projects AGL2004-00194 and AGL2007-61948, co-financed with FEDER), the Spain-Italy Integrated Action HI2007-0228, the European Commission (Thematic Priority 5- Food Quality and Safety, 6th Framework RTD Programme, Contract no. FP6-FOOD-CT-2006-016279) and the Aragón Government (group A03). Acquisition of the HPLC-MS apparatus was cofinanced with FEDER.