Ana G. L. Assunção

Progress in remediation and revegetation of the barren Jales gold mine spoil after in situ treatments

A series of single extractions and short-term plant tests were performed in order to test a variety of inexpensive mineral amendments for the in situ inactivation of trace elements on the fine-grained spoil of the former gold mine of Jales, Portugal. Based on the results of these tests, mesocosms (lysimeters) were constructed and a small-scale semi-field trial was carried out since 1998. The long-term effect of steelshots (SS, iron grit), beringite (B), and municipal compost (C) as spoil amendments was investigated. Vegetation establishment on the treated spoils was successful with Holcus lanatus L. in year 1 and Pinus pinaster Ait. in year 2. Therefore, a detailed monitoring program was implemented for determining the sustainability of trace elements in situ inactivation by C (5%), CB (5% compost combined with 5% beringite), CSS (5% compost combined with 1% steelshots), and CBSS (5% compost combined with 5% beringite and 1% steelshots) treatments (all amendments are expressed by soil dry weight) and of the revegetation. After 3 years, revegetation was excellent in the CSS treatment, and successful for the CBSS. Volunteer plant species became established in treated spoils during year 2. In contrast, the trees planted on the C treated spoil declined from year 2 and some died. In year 3, the trees on the CB treatment started to decline. Arsenic and zinc exposure are suggested to explain the negative effects on pine growth. In agreement with results of single extractions, compost addition in the spoil increased long-term arsenic percolation. Lead leaching was also enhanced. The CBSS and CSS treatments were the most effective for limiting water-soluble As and decreasing long-term metal leaching.

Arabidopsis thaliana transcription factors bZIP19 and bZIP23 regulate the adaptation to zinc deficiency

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.

Revegetation of the acidic, As contaminated Jales mine spoil tips using a combination of spoil amendments and tolerant grasses

A combination of metal immobilising agents and metal tolerant plants has been utilised in order to reduce the environmental impact of the acidic metal contaminated Jales mine spoil tips. The addition of Beringite (a modified aluminosilicate), steel shots (iron bearing material) and organic matter as spoil amendments resulted in changes in arsenate (As) concentrations and pH of spoil material and improved plant growth. The application of Beringite increased both pH and plant available As concentrations. A 4-year follow up of the spoil analysis demonstrated that the effect of the spoil treatments was stable following treatments, however, the effectiveness did not increase any more after 2 years. The use of metal tolerant grasses in combination with spoil treatments resulted in a rapid and effective revegetation of the As contaminated Jales mine spoils. Colonisation and reproduction of Agrostis castellana and Holcus lanatus was most successful when the substrate contained a combination of all three additives. Plant performances could be enhanced by supplementation of a phosphate fertiliser. The rapid reproduction of the two grass species makes them very suitable for revegetation purposes. Agrostis castellana and Holcus lanatus apparently exhibited a level of metal- and As-tolerance sufficient for survival on untreated spoil, but in the first stages of revegetation the use of spoil amendments was found to be essential. Organic matter in combination with Beringite and/or steel shots resulted in decreased As in the aboveground biomass, protecting possible grazers and predators from undesirable levels of As.

Activity of the AtMRP3 promoter in transgenic Arabidopsis thaliana and Nicotiana tabacum plants is increased by cadmium, nickel, arsenic, cobalt and lead but not by zinc and iron.

Characterization of the function, regulation and metal-specificity of metal transporters is one of the basic steps needed for the understanding of transport and accumulation of toxic metals and metalloids by plants. In this work GUS was used as a reporter for monitoring the activity of the promoter of the AtMRP3 gene from Arabidopsis thaliana, a gene encoding an ABC-transporter, expression of which is induced by heavy metals. The AtMRP3 promoter-GUS fusion expression cassette was introduced into the genome of two model plants, A. thaliana and Nicotiana tabacum. The promoter induces GUS activity in the roots as well as in the shoots upon metal exposure. Similar responses of the AtMRP3 promoter to the presence of the selected metals was observed in both plant species. Cadmium, nickel, arsenic, cobalt and lead strongly activated the transcription of the reporter gene, while zinc and iron had no impact. The AtMRP3 promoter thus seems to be a useful new tool in designing plants that can be used for biomonitoring of environmental contaminations.

Regulation of the adaptation to zinc deficiency in plants.

The molecular mechanisms by which plants sense their micronutrient status, and adapt to their environment in order to ensure a sufficient micronutrient supply, are poorly understood. 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 were recently identified. In this mini-review, we highlight recent progress in understanding the adaptation to zinc deficiency in plants and discuss the future challenges to fully unravel its molecular basis.

Model of how plants sense zinc deficiency

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.

Transcriptomic profiling of Arabidopsis gene expression in response to varying micronutrient zinc supply

Deficiency of the micronutrient zinc is a widespread condition in agricultural soils, causing a negative impact on crop quality and yield. Nevertheless, there is an insufficient knowledge on the regulatory and molecular mechanisms underlying the plant response to inadequate zinc nutrition [1]. This information should contribute to the development of plant-based solutions with improved nutrient-use-efficiency traits in crops. Previously, the transcription factors bZIP19 and bZIP23 were identified as essential regulators of the response to zinc deficiency in Arabidopsis thaliana [2]. A microarray experiment comparing gene expression between roots of wild-type and the mutant bzip19 bzip23, exposed to zinc deficiency, led to the identification of differentially expressed genes related with zinc homeostasis, namely its transport and plant internal translocation [2]. Here, we provide the detailed methodology, bioinformatics analysis and quality controls related to the microarray gene expression profiling published by Assunção and co-workers [2]. Most significantly, the present dataset comprises new experimental variables, including analysis of shoot tissue, and zinc sufficiency and excess supply. Thus, it expands from 8 to 42 microarrays hybridizations, which have been deposited at the Gene Expression Omnibus (GEO) under the accession number GSE77286. Overall, it provides a resource for research on the molecular basis and regulatory events of the plant response to zinc supply, emphasizing the importance of Arabidopsis bZIP19 and bZIP23 transcription factors.

Phylogenetic analysis of F-bZIP transcription factors indicates conservation of the zinc deficiency response across land plants

Basic leucine zipper (bZIP) transcription factors control important developmental and physiological processes in plants. In Arabidopsis thaliana, the three gene F-bZIP subfamily has been associated with zinc deficiency and salt stress response. Benefiting from the present abundance of plant genomic data, we performed an evolutionary and structural characterization of plant F-bZIPs. We observed divergence during seed plant evolution, into two groups and inferred different selective pressures for each. Group 1 contains AtbZIP19 and AtbZIP23 and appears more conserved, whereas Group 2, containing AtbZIP24, is more prone to gene loss and expansion events. Transcriptomic and experimental data reinforced AtbZIP19/23 as pivotal regulators of the zinc deficiency response, mostly via the activation of genes from the ZIP metal transporter family, and revealed that they are the main regulatory switch of AtZIP4. A survey of AtZIP4 orthologs promoters across different plant taxa revealed an enrichment of the Zinc Deficiency Response Element (ZDRE) to which both AtbZIP19/23 bind. Overall, our results indicate that while the AtbZIP24 function in the regulation of the salt stress response may be the result of neo-functionalization, the AtbZIP19/23 function in the regulation of the zinc deficiency response may be conserved in land plants (Embryophytes).

Regulation of Micronutrient Homeostasis and Deficiency Response in Plants

Abstract Micronutrients are essential for plant survival despite the low amount needed in comparison to macronutrients. The dynamic range of optimal internal concentrations for micronutrients is thus particularly narrow, and this requires a tight regulation so that its requirements are met under different developmental and environmental conditions. There has been much progress in identifying regulators of the micronutrient metal homeostasis network in plants. These regulators control the expression of genes involved in metal acquisition, mobilization, and storage in response to variations in metal availability, particularly in response to deficiency. Here we present a brief overview on the mechanisms of regulation of iron, copper, and zinc homeostasis and deficiency responses in model plant species. Advances in this area, followed by translational approaches into crops, will likely provide new avenues for breeding crops with improved micronutrient use efficiency and nutritional value.