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 leucinezipper (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 Irtrelated 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.biofortification | zinc homeostasis regulation | plant nutrition | abiotic stress | adaptation Z inc is an essential cofactor for many transcription factors, protein interaction domains, and enzymes in plants (1). Plants are thought to control zinc homeostasis by using a tightly regulated network of zinc status sensors and signal transducers controlling the coordinated expression of proteins involved in zinc acquisition from soil, mobilization between organs and tissues, and sequestration within cellular compartments (2). Although candidate genes for the required proteins such as zinc transporters and chelator biosynthesizing enzymes are found, no regulator of such network was ever identified in plants.Zinc influx facilitators, members of the ZIP family of metal transporters, are thought to play a major role in zinc uptake in plants (3). In Arabidopsis there are 15 ZIP genes (4), with ZIP1, ZIP2, ZIP3, and IRT3 functionally characterized as zinc uptake transporters (3, 5). Gene expression analysis has shown that approximately half of the ZIP genes are induced in response to zinc deficiency (3,(5)(6)(7)(8). The ZIP4 gene in particular is strongly induced upon shortage in zinc supply (3,(6)(7)(8).We focused on the promoter of this zinc-deficiency-responsive gene as the starting point for unraveling the regulation of the zinc homeostasis network in plants. By using DNA fragments of the zincdeficiency-responsive Arabidopsi...

When plants are subjected to high metal exposure, different plant species take different strategies in response to metal-induced stress. Largely, plants can be distinguished in four groups: metal-sensitive species, metal-resistant excluder species, metal-tolerant non-hyperaccumulator species, and metal-hypertolerant hyperaccumulator species, each having different molecular mechanisms to accomplish their resistance/tolerance to metal stress or reduce the negative consequences of metal toxicity. Plant responses to heavy metals are molecularly regulated in a process called metal homeostasis, which also includes regulation of the metal-induced reactive oxygen species (ROS) signaling pathway. ROS generation and signaling plays an important duel role in heavy metal detoxification and tolerance. In this review, we will compare the different molecular mechanisms of nutritional (Zn) and non-nutritional (Cd) metal homeostasis between metal-sensitive and metal-adapted species. We will also include the role of metal-induced ROS signal transduction in this comparison, with the aim to provide a comprehensive overview on how plants cope with Zn/Cd stress at the molecular level.

Summary ZIP transporters (ZRT, IRT‐like proteins) are involved in the transport of iron (Fe), zinc (Zn) and other divalent metal cations. The expression of IRT3, a ZIP transporter, is higher in the Zn/cadmium (Cd) hyperaccumulator Arabidopsis halleri than is that of its ortholog in Arabidopsis thaliana, which implies a positive association of its expression with Zn accumulation in A. halleri. IRT3 genes from both A. halleri and A. thaliana functionally complemented the Zn uptake mutant Spzrt1 in Schizosaccharomyces pombe; and Zn uptake double mutant zrt1zrt2, Fe‐uptake mutant fet3fet4 and conferred Zn and Fe uptake activity in Saccharomyces cerevisiae. By contrast, the manganese (Mn) uptake mutant smf1 phenotypes were not rescued. Insufficient Cd uptake for toxicity was found. Expression of IRT3‐green fluorescent protein (GFP) fusion proteins in Arabidopsis root protoplasts indicated localization of both IRT3 proteins in the plasma membrane. Overexpressing AtIRT3 in A. thaliana led to increased accumulation of Zn in the shoot and Fe in the root of transgenic lines. Therefore, IRT3 functions as a Zn and Fe‐uptake transporter in Arabidopsis.

Populations of Noccaea caerulescens show tremendous differences in their capacity to hyperaccumulate and hypertolerate metals. To explore the differences that could contribute to these traits, we undertook SOLiD high-throughput sequencing of the root transcriptomes of three phenotypically well-characterized N. caerulescens accessions, i.e., Ganges, La Calamine, and Monte Prinzera. Genes with possible contribution to zinc, cadmium, and nickel hyperaccumulation and hypertolerance were predicted. The most significant differences between the accessions were related to metal ion (di-, trivalent inorganic cation) transmembrane transporter activity, iron and calcium ion binding, (inorganic) anion transmembrane transporter activity, and antioxidant activity. Analysis of correlation between the expression profile of each gene and the metal-related characteristics of the accessions disclosed both previously characterized (HMA4, HMA3) and new candidate genes (e.g., for nickel IRT1, ZIP10, and PDF2.3) as possible contributors to the hyperaccumulation/tolerance phenotype. A number of unknown Noccaea-specific transcripts also showed correlation with Zn(2+), Cd(2+), or Ni(2+) hyperaccumulation/tolerance. This study shows that N. caerulescens populations have evolved great diversity in the expression of metal-related genes, facilitating adaptation to various metalliferous soils. The information will be helpful in the development of improved plants for metal phytoremediation.

Noccaea caerulescens is an extremophile plant species belonging to the Brassicaceae family. It has adapted to grow on soils containing high, normally toxic, concentrations of metals such as nickel, zinc, and cadmium. Next to being extremely tolerant to these metals, it is one of the few species known to hyperaccumulate these metals to extremely high concentrations in their aboveground biomass. In order to provide additional molecular resources for this model metal hyperaccumulator species to study and understand the mechanism of adaptation to heavy metal exposure, we aimed to provide a comprehensive database of transcript sequences for N. caerulescens. In this study, 23,830 transcript sequences (isotigs) with an average length of 1025 bp were determined for roots, shoots and inflorescences of N. caerulescens accession “Ganges” by Roche GS-FLEX 454 pyrosequencing. These isotigs were grouped into 20,378 isogroups, representing potential genes. This is a large expansion of the existing N. caerulescens transcriptome set consisting of 3705 unigenes. When translated and compared to a Brassicaceae proteome set, 22,232 (93.2%) of the N. caerulescens isotigs (corresponding to 19,191 isogroups) had a significant match and could be annotated accordingly. Of the remaining sequences, 98 isotigs resembled non-plant sequences and 1386 had no significant similarity to any sequence in the GenBank database. Among the annotated set there were many isotigs with similarity to metal homeostasis genes or genes for glucosinolate biosynthesis. Only for transcripts similar to Metallothionein3 (MT3), clear evidence for an additional copy was found. This comprehensive set of transcripts is expected to further contribute to the discovery of mechanisms used by N. caerulescens to adapt to heavy metal exposure.

Prompt regulation of transition metal transporters is crucial for plant zinc homeostasis. NcZNT1 is one of such transporters, found in the metal hyperaccumulator Brassicaceae species Noccaea caerulescens. It is orthologous to AtZIP4 from Arabidopsis thaliana, an important actor in Zn homeostasis. We examined if the NcZNT1 function contributes to the metal hyperaccumulation of N. caerulescens. NcZNT1 was found to be a plasma-membrane located metal transporter. Constitutive overexpression of NcZNT1 in A. thaliana conferred enhanced tolerance to exposure to excess Zn and Cd supply, as well as increased accumulation of Zn and Cd and induction of the Fe deficiency response, when compared to non-transformed wild-type plants. Promoters of both genes were induced by Zn deficiency in roots and shoots of A. thaliana. In A. thaliana, the AtZIP4 and NcZNT1 promoters were mainly active in cortex, endodermis and pericycle cells under Zn deficient conditions. In N. caerulescens, the promoters were active in the same tissues, though the activity of the NcZNT1 promoter was higher and not limited to Zn deficient conditions. Common cis elements were identified in both promoters by 5’ deletion analysis. These correspond to the previously determined Zinc Deficiency Responsive Elements found in A. thaliana to interact with two redundantly acting transcription factors, bZIP19 and bZIP23, controlling the Zn deficiency response. In conclusion, these results suggest that NcZNT1 is an important factor in contributing to Zn and Cd hyperaccumulation in N. caerulescens. Differences in cis- and trans-regulators are likely to account for the differences in expression between A. thaliana and N. caerulescens. The high, constitutive NcZNT1 expression in the stele of N. caerulescens roots implicates its involvement in long distance root-to-shoot metal transport by maintaining a Zn/Cd influx into cells responsible for xylem loading.

In this study we engineered yeast cells armed for heavy metal accumulation by targeting plant metallothioneins to the inner face of the yeast plasma membrane. Metallothioneins (MTs) are cysteine-rich proteins involved in the buffering of excess metal ions, especially Cu(I), Zn(II) or Cd(II). The cDNAs of seven Arabidopsis thaliana MTs (AtMT1a, AtMT1c, AtMT2a, AtMT2b, AtMT3, AtMT4a and AtMT4b) and four Noccaea caerulescens MTs (NcMT1, NcMT2a, NcMT2b and NcMT3) were each translationally fused to the C-terminus of a myristoylation green fluorescent protein variant (myrGFP) and expressed in Saccharomyces cerevisiae cells. The myrGFP cassette introduced a yeast myristoylation sequence which allowed directional targeting to the cytosolic face of the plasma membrane along with direct monitoring of the intracellular localization of the recombinant protein by fluorescence microscopy. The yeast strains expressing plant MTs were investigated against an array of heavy metals in order to identify strains which exhibit the (hyper)accumulation phenotype without developing toxicity symptoms. Among the transgenic strains which could accumulate Cu(II), Zn(II) or Cd(II), but also non-canonical metal ions, such as Co(II), Mn(II) or Ni(II), myrGFP-NcMT3 qualified as the best candidate for bioremediation applications, thanks to the robust growth accompanied by significant accumulative capacity.

Cnaphalocrocis medinalis is a major insect pest of rice in Asia. A few defensive enzymes were reported to show higher activities in a resistant rice line (Qingliu) than in a susceptible rice line (TN1) upon leaffolder infestation. However, the overall molecular regulation of the rice defense response against leaffolder herbivory is unknown. Here, differential proteomic analysis by SWATH-MS was performed to identify differentially expressed proteins between the two rice varieties, Qingliu and TN1, at four time points of leaffolder herbivory, 0, 6, 24, and 72 h. Gene Ontology (GO) enrichment of the differentially expressed proteins indicated overrepresentation of (1) photosynthesis, (2) amino acid and derivative metabolic process, and (3) secondary metabolic process. Phenylalanine ammonia lyase and chalcone synthase, which catalyze flavonoid biosynthesis, and lipoxygenase, which catalyzes jasmonic acid biosynthesis, exhibited higher expression in Qingliu than in TN1 even before insect herbivory. Momentary activation of the light reaction and Calvin cycle was detected in Qingliu at 6 h and 24 h of insect herbivory, respectively. At 72 h of insect herbivory, amino acid biosynthesis and glutathionemediated antioxidation were activated in Qingliu. A defense response involving jasmonic acid signaling, carbon remobilization, and the production of flavonoids and glutathione could underlie the resistance of Qingliu to leaffolder. Rice production in Asia is affected by a harmful insect pest, Cnaphalocrocis medinalis (Guenée) (Lepidoptera: Pyralidae), also known as the rice leaffolder 1. Leaffolder caterpillars feed on mesophyll tissues, interfering with photosynthesis and then reducing yield 2. The affected leaf blades appear white, so heavily infested fields may have 'scorched'-looking patches. This insect herbivore can complete three life cycles during each rice cropping season in Taiwan 3,4. The application of chemical insecticides is the main method used to control pest populations in rice fields 5. However, this method of pest control is rendered less effective because the caterpillars build a feeding chamber by folding a leaf longitudinally with silk, and the chamber indirectly protects the pests from the chemical spray. When plants are infested by insect pests, they can differentiate diverse types of insects based on the insect elicitors or the nature of the damage caused by the insects 6. The plants immediately activate various defensive signaling pathways, including those associated with mitogen-activated protein kinase (MAPK) and phytohormones, such as jasmonic acid (JA), salicylic acid (SA) and ethylene (ET) 7. Chewing insects are generally known to trigger JA signaling pathways, while phloem-feeding insects trigger the SA signaling pathway 8. The JA and SA signaling pathways also participate in antagonistic crosstalk in plant-insect herbivory interactions 8. Because the signaling pathways triggered by different herbivorous insects vary, the production of downstream defensive proteins and secondary metabolites is...