Assessing Tolerance to Heavy-Metal Stress in Arabidopsis thaliana Seedlings

The deposited book chapter is a post-print version and has been submitted to peer review.The deposited book chapter version contains attached the supplementary materials within the pdf.This publication hasn't any creative commons license associated.The deposited book chapter is part of the book series: "Environmental Responses in Plants: Methods and Protocols" (pp.197-208) published by Springer.Heavy-metal soil contamination is one of the major abiotic stress factors that, by negatively affecting plant growth and development, severely limit agricultural productivity worldwide. Plants have evolved various tolerance and detoxification strategies in order to cope with heavy-metal toxicity while ensuring adequate supply of essential micronutrients at the whole-plant as well as cellular levels. Genetic studies in the model plant Arabidopsis thaliana have been instrumental in elucidating such mechanisms. The root assay constitutes a very powerful and simple method to assess heavy-metal stress tolerance in Arabidopsis seedlings. It allows the simultaneous determination of all the standard growth parameters affected by heavy-metal stress (primary root elongation, lateral root development, shoot biomass, and chlorophyll content) in a single experiment. Additionally, this protocol emphasizes the tips and tricks that become particularly useful when quantifying subtle alterations in tolerance to a given heavy-metal stress, when simultaneously pursuing a large number of plant lines, or when testing sensitivity to a wide range of heavy metals for a single line.Fundação para a Ciência e a Tecnologia grants: (EXPL/AGR-PRO/1013/2013, SFRH/BPD/44640/2008); GREEN-it "Bioresources for Sustainability": (UID/Multi/04551/2013).info:eu-repo/semantics/publishedVersio

Genetic dissection of shoot development in maize

The isolation of genes affecting plant shoot formation is an important prerequisite for understanding the logic of plant development as well as for manipulating plant architecture. To this aim, maize transpositional mutagenesis has been adopted in our laboratory and has led to the isolation of different developmental mutants that are currently under study. Two of them, shootmeristemless (sml) and fused leaves (fdl), are presently described. Their locations, on the long arm of chromosome 10 and chromosome 7 respectively, have been established by traditional B-A translocation mapping and subsequently by linkage analysis with visible, as well as molecular, markers. The sml gene is a recessive mutation affecting shoot apical meristem maintenance and lateral organ formation. Its introgression in different genetic backgrounds has highlighted the epistatic interaction between sml and the unlinked distorted growth (dgr) locus. Seeds homozygous for both sml and dgr loci have a shootless phenotype whereas Dgr/-sml/sml seeds produce plants with altered fillotaxis and abnormal leaf morphogenesis. Microscopy analysis of young dgr primordia will be presented showing that the mutation does not interfere with epidermis formation although cell shape is less regular than in the wild-type. The fdl mutation confers a pleiotropic phenotype to the maize plant, which is specifically confined to the juvenile phase. The lack of a regular coleoptile opening that in normal development occurs after germination, is one of the fdl specific traits that we are presently investigating, for its putative relationship with the programmed cell death (PCD) process. We will present preliminary data on the cloning of the fdl gene along with a description of the strategy we have undertaken for the isolation of novel alleles. They will constitute proof for the gene identity and will provide new genetic material suitable for functional studies

Long-term field metal extraction by pelargonium:phytoextraction efficiency in relation to plant maturity

The long length of periods required for effective soil remediation via phytoextraction constitutes a weak point that reduces its industrial use. However, these calculated periods are mainly based on short-term and/or hydroponic controlled experiments. Moreover, only a few studies concern more than one metal, although soils are scarcely polluted by only one element.In this scientific context, the phytoextraction of metals and metalloids (Pb, Cd, Zn, Cu,and As) by Pelargonium was measured after a long-term field experiment. Both bulk and rhizosphere soils were analyzed in order to determine the mechanisms involved in soil-root transfer. First, a strong increase in lead phytoextraction was observed with plant maturity, significantly reducing the length of the period required for remediation. Rhizosphere Pb, Zn, Cu, Cd, and As accumulation was observed (compared to bulk soil), indicating metal mobilization by the plant, perhaps in relation to root activity. Moreover, metal phytoextraction and translocation were found to be a function of the metals’ nature. These results, taken altogether, suggest that Pelargonium could be used as a multi-metal hyperaccumulator under multi-metal soil contamination conditions, and they also provide an interesting insight for improving field phytoextraction remediation in terms of the length of time required, promoting this biological technique

Karyopherins: potential biological elements involved in the delayed graft function in renal transplant recipients.

Background: Immediately after renal transplantation, patients experience rapid and significant improvement of their clinical conditions and undergo considerable systemic and cellular modifications. However, some patients present a slow recovery of the renal function commonly defined as delayed graft function (DGF). Although clinically well characterized, the molecular mechanisms underlying this condition are not totally defined, thus, we are currently missing specific clinical markers to predict and to make early diagnosis of this event.Methods: We investigated, using a pathway analysis approach, the transcriptomic profile of peripheral blood mononuclear cells (PBMC) from renal transplant recipients with DGF and with early graft function (EGF), before (T0) and 24 hours (T24) after transplantation.Results: Bioinformatics/statistical analysis showed that 15 pathways (8 up-regulated and 7 down-regulated) and 11 pathways (5 up-regulated and 6 down-regulated) were able to identify DGF patients at T0 and T24, respectively. Interestingly, the most up-regulated pathway at both time points was NLS-bearing substrate import into nucleus, which includes genes encoding for several subtypes of karyopherins, a group of proteins involved in nucleocytoplasmic transport. Signal transducers and activators of transcription (STAT) utilize karyopherins-alpha (KPNA) for their passage from cytoplasm into the nucleus. In vitro functional analysis demonstrated that in PBMCs of DGF patients, there was a significant KPNA-mediated nuclear translocation of the phosphorylated form of STAT3 (pSTAT3) after short-time stimulation (2 and 5 minutes) with interleukin-6.Conclusions: Our study suggests the involvement, immediately before transplantation, of karyopherin-mediated nuclear transport in the onset and development of DGF. Additionally, it reveals that karyopherins could be good candidates as potential DGF predictive clinical biomarkers and targets for pharmacological interventions in renal transplantation. However, because of the low number of patients analyzed and some methodological limitations, additional studies are needed to validate and to better address these points

Behavior and Impact of Zirconium in the Soil–Plant System: Plant Uptake and Phytotoxicity

Because of the large number of sites they pollute, toxic metals that contaminate terrestrial ecosystems are increasingly of environmental and sanitary concern (Uzu et al. 2010, 2011; Shahid et al. 2011a, b, 2012a). Among such metals is zirconium (Zr), which has the atomic number 40 and is a transition metal that resembles titanium in physical and chemical properties (Zaccone et al. 2008). Zr is widely used in many chemical industry processes and in nuclear reactors (Sandoval et al. 2011; Kamal et al. 2011), owing to its useful properties like hardness, corrosion-resistance and permeable to neutrons (Mushtaq 2012). Hence, the recent increased use of Zr by industry, and the occurrence of the Chernobyl and Fukashima catastrophe have enhanced environmental levels in soil and waters (Yirchenko and Agapkina 1993; Mosulishvili et al. 1994 ; Kruglov et al. 1996)

\u3ci\u3eSenecio Conrathii\u3c/i\u3e N.E.Br. (Asteraceae), a New Hyperaccumulator of Nickel from Serpentinite Outcrops of the Barberton Greenstone Belt, South Africa

Five nickel hyperaccumulators belonging to the Asteraceae are known from ultramafic outcrops in South Africa. Phytoremediation applications of the known hyperaccumulators in the Asteraceae, such as the indigenous Berkheya coddii Roessler, are well reported and necessitate further exploration to find additional species with such traits. This study targeted the most frequently occurring species of the Asteraceae on eight randomly selected serpentinite outcrops of the Barberton Greenstone Belt. Twenty species were sampled, including 12 that were tested for nickel accumulation for the first time. Although the majority of the species were excluders, the known hyperaccumulators Berkheya nivea N.E.Br. and B. zeyheri (Sond. & Harv.) Oliv. & Hiern subsp. rehmannii (Thell.) Roessler var. rogersiana (Thell.) Roessler hyperaccumulated nickel in the leaves at expected levels. A new hyperaccumulator of nickel was discovered, Senecio conrathii N.E.Br., which accumulated the element in its leaves at 1695 ± 637 µg g−1 on soil with a total and exchangeable nickel content of 503 mg kg−1 and 0.095 µg g−1, respectively. This makes it the third known species in the Senecioneae of South Africa to hyperaccumulate nickel after Senecio anomalochrous Hilliard and Senecio coronatus (Thunb.) Harv., albeit it being a weak accumulator compared with the latter. Seven tribes in the Asteraceae have now been screened for hyperaccumulation in South Africa, with hyperaccumulators only recorded for the Arctoteae and Senecioneae. This suggests that further exploration for hyperaccumulators should focus on these tribes as they comprise all six species (of 68 Asteraceae taxa screened thus far) to hyperaccumulate nickel

A preliminary study of the role of nickel in enhancing flowering of the nickel hyperaccumulating plant Alyssum inflatum Nyár. (Brassicaceae)

Alyssum inflatum is a native of serpentine soils and is able to hyperaccumulate nickel (Ni), but the importance of Ni to reproduction in the species is unknown. We investigated if reproductive fitness is enhanced by Ni in the growth medium, and included a treatment involving a relatively high level of Mg to provide a comparison with elevated levels of another metal. Seedlings were grown in a modified Hoagland solution culture in an inertmediumof Perlite andwere treatedwith Ni (100 μM), a high concentration ofMg (5mM), or under control conditions (solution culture without Ni or the addition of high Mg) for 14 months.We documented survival, as well as the proportion of individuals that flowered.We also quantified flower production as an indicator of plant fitness. Survival was not affected by treatment (87–90% for all treatments), but significantly more Ni-treated plants (63%) flowered compared with Mg-treated (19%) or control plants (12%). In addition, inflorescences per plant, inflorescence length, and number of open flowers per inflorescence were all significantly greater for Nitreated plants relative to plants fromthe other treatments. Although high levels of Ni are not essential for growth and reproduction of the species, we suggest that Ni stimulates flowering in A. inflatum and may result in greater fitness for the species on serpentine soils

Genetic Structure of Modern Durum Wheat Cultivars and Mediterranean Landraces Matches with Their Agronomic Performance

A collection of 172 durum wheat landraces from 21 Mediterranean countries and 20 modern cultivars were phenotyped in 6 environments for 14 traits including phenology, biomass, yield and yield components. The genetic structure of the collection was ascertained with 44 simple sequence repeat markers that identified 448 alleles, 226 of them with a frequency lower than 5%, and 10 alleles per locus on average. In the modern cultivars all the alleles were fixed in 59% of the markers. Total genetic diversity was HT = 0.7080 and the genetic differentiation value was GST = 0.1730. STRUCTURE software allocated 90.1% of the accessions in five subpopulations, one including all modern cultivars, and the four containing landrace related to their geographic origin: eastern Mediterranean, eastern Balkans and Turkey, western Balkans and Egypt, and western Mediterranean. Mean yield of subpopulations ranged from 2.6 t ha-1 for the western Balkan and Egyptian landraces to 4.0 t ha-1 for modern cultivars, with the remaining three subpopulations showing similar values of 3.1 t ha-1. Modern cultivars had the highest number of grains m-2 and harvest index, and the shortest cycle length. The diversity was lowest in modern cultivars (HT = 0.4835) and highest in landraces from the western Balkans and Egypt (HT = 0.6979). Genetic diversity and AMOVA indicated that variability between subpopulations was much lower (17%) than variability within them (83%), though all subpopulations had similar biomass values in all growth stages. A dendrogram based on simple sequence repeat data matched with the clusters obtained by STRUCTURE, improving this classification for some accessions that have a large admixture. landraces included in the subpopulation from the eastern Balkans and Turkey were separated into two branches in the dendrogram drawn with phenotypic data, suggesting a different origin for the landraces collected in Serbia and Macedonia. The current study shows a reliable relationship between genetic and phenotypic population structures, and the connection of both with the geographic origin of the landraces.The research was funded by the Ministerio de Economía y competitividad project AGL-2006-09226-C02-01, and Dr. Jose Miguel Soriano is funded by Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (http://www.mineco.gob.es/)

Does Speciation between Arabidopsis halleri and Arabidopsis lyrata Coincide with Major Changes in a Molecular Target of Adaptation?

Ever since Darwin proposed natural selection as the driving force for the origin of species, the role of adaptive processes in speciation has remained controversial. In particular, a largely unsolved issue is whether key divergent ecological adaptations are associated with speciation events or evolve secondarily within sister species after the split. The plant Arabidopsis halleri is one of the few species able to colonize soils highly enriched in zinc and cadmium. Recent advances in the molecular genetics of adaptation show that the physiology of this derived ecological trait involves copy number expansions of the AhHMA4 gene, for which orthologs are found in single copy in the closely related A. lyrata and the outgroup A. thaliana. To gain insight into the speciation process, we ask whether adaptive molecular changes at this candidate gene were contemporary with important stages of the speciation process. We first inferred the scenario and timescale of speciation by comparing patterns of variation across the genomic backgrounds of A. halleri and A. lyrata. Then, we estimated the timing of the first duplication of AhHMA4 in A. halleri. Our analysis suggests that the historical split between the two species closely coincides with major changes in this molecular target of adaptation in the A. halleri lineage. These results clearly indicate that these changes evolved in A. halleri well before industrial activities fostered the spread of Zn- and Cd-polluted areas, and suggest that adaptive processes related to heavy-metal homeostasis played a major role in the speciation process