Ectopic expression of nicotianamine synthase genes results in improved iron accumulation and increased nickel tolerance in transgenic tobacco

Heavy metals are essential for basic cellular processes but toxic in higher concentrations. This requires the precise control of their intracellular concentrations, a process known as homeostasis. The metal-chelating, non-proteinogenous amino acid nicotianamine (NA) is a key component of plant metal assimilation and homeostasis. Its precise function is still unknown. Therefore, this article aims to contribute new information on the in vivo function of NA and to evaluate its potential use for plant nutrition and crop fortification. For this purpose, a nicotianamine synthase gene of Arabidopsis thaliana was ectopically expressed in transgenic tobacco plants. The presence of extra copies of the nicotianamine synthase gene co-segregated with up to 10-fold elevated levels of NA in comparison with wild type. The increased NA level led to: (a) a significantly increased iron level in leaves of adult plants; (b) the accumulation of zinc and manganese, but not copper; (c) an improvement of the iron use efficiency in adult plants grown under iron limitation; and (d) an enhanced tolerance against up to 1 m m nickel. Taken together, the data predict that NA may be a useful tool for improved plant nutrition on adverse soils and possibly for enhanced nutritional value of leaf and seed crops.     

Increased nicotianamine biosynthesis confers enhanced tolerance of high levels of metals, in particular nickel, to plants

Nicotianamine, a plant-derived chelator of metals, is produced by the trimerization of S-adenosylmethionine catalyzed by nicotianamine synthase. We established transgenic Arabidopsis and tobacco plants that constitutively overexpress the barley nicotianamine synthase gene. Nicotianamine synthase overexpression resulted in increased biosynthesis of nicotianamine in transgenic plants, which conferred enhanced tolerance of high levels of metals, particularly nickel, to plants. Promoter activities of four nicotianamine synthase genes in Arabidopsis were all increased in response to excess nickel, suggesting that nicotianamine plays an important role in the detoxification of nickel in plants. Furthermore, transgenic tobacco plants with a high level of nicotianamine grew well in a nickel-enriched serpentine soil without developing any symptoms of nickel toxicity. Our results indicate that nicotianamine plays a critical role in metal detoxification, and this can be a powerful tool for use in phytoremediation.     

Root-to-shoot long-distance circulation of nicotianamine and nicotianamine-nickel chelates in the metal hyperaccumulator Thlaspi caerulescens

Plant metal hyperaccumulator species are widely used as models to unravel the heavy metal tolerance and hyperaccumulation mechanisms. Thlaspi caerulescens is capable of tolerating and hyperaccumulating Zn, Cd, and Ni. A search for factors involved in the cellular tolerance to Ni, based on yeast screens, led to isolation of a cDNA encoding a functional nicotianamine (NA) synthase (NAS). The T. caerulescens genome appears to contain a single copy of the NAS gene named TcNAS whose expression is restricted to the leaves. The analysis of dose-response and time-course Ni treatments have revealed that the exposure to Ni triggers the accumulation of NA in the roots. Because neither TcNAS expression nor NAS activity were detected in the roots, the NA accumulation in roots is most probably the result of its translocation from the leaves. Once in the roots, NA, together with Ni, is subsequently found in the xylem, for redirection to the aerial parts. Using liquid chromatography coupled to inductively coupled plasma or electrospray ionization mass spectrometry, it has been shown that part of the Ni is translocated as a stable Ni-NA complex in the xylem sap. This circulation of NA, Ni, and NA-Ni chelates is absent in the non-tolerant non-hyperaccumulator related species T. arvense. Taken together, the results provide direct physiological and chemical evidence for NA and NA-heavy metal complex translocation in a hyperaccumulator species.     

Fe homeostasis in plant cells: Does nicotianamine play multiple roles in the regulation of cytoplasmic Fe concentration?

The cellular and intracellular localization of the non-proteogenic amino acid nicotianamine (NA) in leaves and root elongation zones was immunochemically investigated in pea (Pisum sativum L.) and tomato (Lycopersicon esculentum Mill.) plants grown under various iron regimes and in three mutants defective in the regulation of iron uptake. Strongest immunostaining was observed in the over-accumulating pea mutants brz and dgl, and in iron-loaded wild-type plants. Fe concentration and NA level paralleled staining intensity, indicating that NA synthesis is induced by high iron availability. While label was mainly present in the cytoplasm under normal (10 microM) Fe supply and under Fe deprivation, most of the labeling was present in the vacuole in iron-loaded plants. This pattern resembled the distribution of NA in Fe over-accumulating mutants, indicating the possible importance of vacuolar sequestration in the detoxification of excess Fe. Based on the dependence of the cellular distribution of NA on the iron nutritional status of the plant, a possible role of NA in buffering free Fe in root and leaf cells was inferred. We show here for the first time that the NA concentration is increased in response to iron overload, indicating that, besides other classes of intracellular metal-binding ligands, NA may play an essential role in iron tolerance.     

The isochorismate pathway is negatively regulated by salicylic acid signaling in O<Subscript>3</Subscript>-exposed <Emphasis Type="Italic">Arabidopsis</Emphasis>

Phytochelatins (PCs) are heavy metal binding peptides that play an important role in sequestration and detoxification of heavy metals in plants. In this study, our goal was to develop transgenic plants with increased tolerance for and accumulation of heavy metals from soil by expressing an Arabidopsis thaliana AtPCS1 gene, encoding phytochelatin synthase (PCS), in Indian mustard (Brassica juncea L.). A 35S promoter fused to a FLAG–tagged AtPCS1 cDNA was expressed in Indian mustard, and transgenic lines, designated pc lines, were evaluated for tolerance to and accumulation of Cd and Zn. Transgenic plants with moderate AtPCS1 expression levels showed significantly higher tolerance to Cd and Zn stress, but accumulated significantly less Cd and Zn than wild type plants in both shoot and root tissues. However, transgenic plants with highest expression of the transgene did not exhibit enhanced Cd and Zn tolerance. Shoots of Cd-treated pc plants had significantly higher levels of phytochelatins and thiols than wild-type plants. Significantly lower concentrations of gluthatione in Cd-treated shoot and root tissues of transgenic plants were observed. Moderate expression levels of phytochelatin synthase improved the ability of Indian mustard to tolerate certain levels of heavy metals, but at the same time did not increase the accumulation potential for Cd and Zn.     

Bio-available zinc in rice seeds is increased by activation tagging of nicotianamine synthase

We generated rice lines with increased content of nicotianamine (NA), a key ligand for metal transport and homeostasis. This was accomplished by activation tagging of rice nicotianamine synthase 2 (OsNAS2). Enhanced expression of the gene resulted in elevated NA levels, greater Zn accumulations and improved plant tolerance to a Zn deficiency. Expression of Zn-uptake genes and those for the biosynthesis of phytosiderophores (PS) were increased in transgenic plants. This suggests that the higher amount of NA led to greater exudation of PS from the roots, as well as stimulated Zn uptake, translocation and seed-loading. In the endosperm, the OsNAS2 activation-tagged line contained up to 20-fold more NA and 2.7-fold more zinc. Liquid chromatography combined with inductively coupled plasma mass spectrometry revealed that the total content of zinc complexed with NA and 2'-deoxymugineic acid was increased 16-fold. Mice fed with OsNAS2-D1 seeds recovered more rapidly from a zinc deficiency than did control mice receiving WT seeds. These results demonstrate that the level of bio-available zinc in rice grains can be enhanced significantly by activation tagging of OsNAS2.     

Nicotianamine synthase: Gene isolation,gene transfer and application for the manipulation of plant iron assimilation

Basic cellular processes such as electron transport in photosynthesis and respiration require the precise control of iron homeostasis. To mobilise iron, plants have evolved at least two different strategies. The non-proteinogenic amino acid nicotianamine is an essential component of both pathways.We briefly review the characterisation of the nicotianamine synthase as a member of a novel class of enzymes, the cloning of the corresponding gene coding sequences of barley, Arabidopsis and tomato as well as the molecular basis of the chloronerva mutant exhibiting severe defects in the regulation of iron metabolism.Further, we report on current experiments aiming to the application of various NAS-genes to manipulate iron assimilation in model and crop plants using transgenic sense and antisense approaches.     

Genetic analysis of two tomato mutants affected in the regulation of iron metabolism

Iron is one of the most important micronutrients for plants. Like other organisms, plants have developed active mechanisms for the acquisition of sufficient iron from the soil. Nevertheless, very little is known about the genetic mechanisms that control the active uptake. In tomato, two spontaneously derived mutants are available, which are defective in key steps that control this process. The recessive mutationchloronerva (chln) affects a gene which controls the synthesis of the non-protein amino acid nicotianamine (NA), a key component in the iron physiology of plants. The root system of the recessive mutantfer is unable to induce any of the characteristic responses to iron deficiency and iron uptake is thus completely blocked. We present a characterization of the double mutant, showing that thefer gene is epistatic over thechln gene and thus very likely to be one of the major genetic elements controlling iron physiology in tomato. In order to gain access to these two genes at the molecular level, both mutants were precisely mapped onto the high density RFLP map of tomato. Thechln gene is located on chromosome 1 and thefer gene is on chromosome 6 of tomato. Using this high-resolution map, a chromosome walk has been started to isolate thefer gene by map-based cloning. The isolation of thefer gene will provide new insights into the molecular mechanisms of iron uptake control in plants.     

Ethephon mitigates nickel stress by modulating antioxidant system,glyoxalase system and proline metabolism in Indian mustard

The role of ethylene (through application of ethephon) in the regulation of nickel (Ni) stress tolerance was investigated in this study. Ethephon at concentration of 200 µl l^?1 was applied to mustard (Brassica juncea) plants grown without and with 200 mg kg^?1 soil Ni to study the increased growth traits, biochemical attributes, photosynthetic efficiency, nutrients content, activities of antioxidants such as superoxide dismutase, ascorbate peroxidase, glutathione reductase, and glutathione peroxidase, glyoxalase systems and enhanced the proline metabolism. In the absence of ethephon, Ni increased oxidative stress with a concomitant decrease in photosynthesis, growth and nutrients content. However, application of ethephon positively increased growth traits, photosynthetic parameters, nutrients content and also elevated the generation of antioxidants enzymes and glyoxalase systems, proline production to combat oxidative stress. Plants water relations and cellular homeostasis were maintained through increased photosynthetic efficiency and proline production. This signifies the role of ethylene in mediating Ni tolerance via regulating proline production and photosynthetic capacity. Ethephon can be used as an exogenous supplement on plants to confer Ni tolerance. The results can be exploited to develop tolerance in plants via gene editing technology encoding enzymes responsible for proline synthesis, antioxidant defence, glyoxalase systems and photosynthetic effectiveness.

     

Overexpression of a <Emphasis Type="Italic">Panax ginseng</Emphasis> tonoplast aquaporin alters salt tolerance,drought tolerance and cold acclimation ability in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> plants

Water movement across cellular membranes is regulated largely by a family of water channel proteins called aquaporins (AQPs). Since several abiotic stresses such as, drought, salinity and freezing, manifest themselves via altering water status of plant cells and are linked by the fact that they all result in cellular dehydration, we overexpressed an AQP (tonoplast intrinsic protein) from Panax ginseng, PgTIP1, in transgenic Arabidopsis thaliana plants to test its role in plant’s response to drought, salinity and cold acclimation (induced freezing tolerance). Under favorable conditions, PgTIP1 overexpression significantly increased plant growth as determined by the biomass production, and leaf and root morphology. PgTIP1 overexpression had beneficial effect on salt-stress tolerance as indicated by superior growth status and seed germination of transgenic plants under salt stress; shoots of salt-stressed transgenic plants also accumulated greater amounts of Na⁺ compared to wild-type plants. Whereas PgTIP1 overexpression diminished the water-deficit tolerance of plants grown in shallow (10 cm deep) pots, the transgenic plants were significantly more tolerant to water stress when grown in 45 cm deep pots. The rationale for this contrasting response, apparently, comes from the differences in the root morphology and leaf water channel activity (speed of dehydration/rehydration) between the transgenic and wild-type plants. Plants overexpressed with PgTIP1 exhibited lower (relative to wild-type control) cold acclimation ability; however, this response was independent of cold-regulated gene expression. Our results demonstrate a significant function of PgTIP1 in growth and development of plant cells, and suggest that the water movement across tonoplast (via AQP) represents a rate-limiting factor for plant vigor under favorable growth conditions and also significantly affect responses of plant to drought, salt and cold stresses.     

The Effect of Paraquat and Hypothermia on Norflurason-Resistant Mutants of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and Derived Cell Cultures

A tolerance to paraquat (PQ) of plants and cell cultures of Arabidopsis thaliana mutants, nfz18 and nfz24, obtained by chemical mutagenesis and selected by their tolerance to norflurason was demonstrated. This tolerance to PQ was manifested in less active peroxidation of lipids (POL), which was assessed by the content of thiobarbituric acid-reactive substances, and in a less degree of plant and callus damages, which was accompanied by a higher activity of superoxide dismutase and other antioxidant enzymes. A capability of norflurason-tolerant mutants to cross-adaptation toward PQ and activation of antioxidant enzymes indicate a genetically determined activation of the antioxidant systems, resulting in improved mutant tolerance to these inducers of oxidative stress. The nfz24 mutant was much more sensitive to hypothermia than wild-type plants and nfz18 mutants, which was expressed in a higher level of POL in plants and calluses and in a more rapid decrease in the suspension cell viability of this mutant. A similarity in the responses of plants and derived heterotrophic cultures to PQ and hypothermia indicates that, in these A. thaliana mutants, adaptation to these types of stresses occurs mainly at the cellular level. Possible reasons of increased sensitivity to hypothermia of the nfz24 mutant, which was more tolerant to the inducers of oxidative stress, PQ and norflurason, are discussed.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 3, 2005, pp. 421–429.Original Russian Text Copyright © 2005 by Volkova, Burgutin, Soldatova, Ezhova, Lapshin.     

Activation of Rice <Emphasis Type="Italic">nicotianamine synthase 2</Emphasis> (<Emphasis Type="Italic">OsNAS2</Emphasis>) enhances iron availability for biofortification

Because micronutrients in human diets ultimately come from plant sources, malnutrition of essential minerals is a significant public health concern. By increasing the expression of nicotianamine synthase (NAS), we fortified the level of bioavailable iron in rice seeds. Activation of iron deficiency-inducible OsNAS2 resulted in a rise in Fe content (3.0-fold) in mature seeds. Its ectopic expression also increased that content. Enhanced expression led to higher tolerance of Fe deficiency and better growth under elevated pH. Mice fed with OsNAS2-D1 seeds recovered more rapidly from anemia, indicating that bioavailable Fe contents were improved by this increase in OsNAS2 expression.     

Two rice cytosolic ascorbate peroxidases differentially improve salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

In order to determine the different roles of rice (Oryza sativa L.) cytosolic ascorbate peroxidases (OsAPXa and OsAPXb, GenBank accession nos. D45423 and AB053297, respectively) under salt stress, transgenic Arabidopsis plants over-expressing OsAPXa or OsAPXb were generated, and they all exhibited increased tolerance to salt stress compared to wild-type plants. Moreover, transgenic lines over-expressing OsAPXb showed higher salt tolerance than OsAPXa transgenic lines as indicated by root length and total chlorophyll content. In addition to ascorbate peroxidase (APX) activity, antioxidant enzyme activities of catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR), which are also involved in the salt tolerance process, and the content of H₂O₂ were also assayed in both transgenic and wild-type plants. The results showed that the overproduction of OsAPXb enhanced and maintained APX activity to a much higher degree than OsAPXa in transgenic Arabidopsis during treatment with different concentrations of NaCl, enhanced the active oxygen scavenging system, and protected plants from salt stress by equilibrating H₂O₂ metabolism. Our findings suggest that the rice cytosolic OsAPXb gene has a more functional role than OsAPXa in the improvement of salt tolerance in transgenic plants. Zhenqiang Lu and Dali Liu contributed equally.     

Metabolic engineering of bread wheat improves grain iron concentration and bioavailability

Bread wheat (Triticum aestivum L.) is cultivated on more land than any other crop and produces a fifth of the calories consumed by humans. Wheat endosperm is rich in starch yet contains low concentrations of dietary iron (Fe) and zinc (Zn). Biofortification is a micronutrient intervention aimed at increasing the density and bioavailability of essential vitamins and minerals in staple crops; Fe biofortification of wheat has proved challenging. In this study we employed constitutive expression (CE) of the rice (Oryza sativa L.) nicotianamine synthase 2 (OsNAS2) gene in bread wheat to up‐regulate biosynthesis of two low molecular weight metal chelators – nicotianamine (NA) and 2′‐deoxymugineic acid (DMA) – that play key roles in metal transport and nutrition. The CE‐OsNAS2 plants accumulated higher concentrations of grain Fe, Zn, NA and DMA and synchrotron X‐ray fluorescence microscopy (XFM) revealed enhanced localization of Fe and Zn in endosperm and crease tissues, respectively. Iron bioavailability was increased in white flour milled from field‐grown CE‐OsNAS2 grain and positively correlated with NA and DMA concentrations.     

The Expression of a Barley HνNAS1 Nicotianamine Synthase Gene Promoter-gus Fusion Gene in Transgenic Tobacco is Induced by Fe-deficiency in Roots

Nicotianamine (NA) is a precursor for mugineic acid-family phytosiderophores, which are a critical component of the Fe aquisition process in graminaceous plants. In addition, nicotianamine synthase (NAS) is strongly induced in these plants by Fe deficiency. NA is essential for Fe metabolism also in dicots, but NAS is not induced by Fe deficiency. We introduced a barley HνNAS1 promoter-gus fusion gene into tobacco. GUS activity was induced in the roots of these plants by Fe deficiency, and was constitutively expressed at a low level in their leaves.     

Isolation of a cDNA clone (PcSrp) encoding serine-rich-protein from Porteresia coarctata T. and its expression in yeast and finger millet (Eleusine coracana L.) affording salt tolerance

A 1.4 Kb cDNA clone encoding a serine-rich protein has been isolated from the cDNA library of salt stressed roots of Porteresia coarctata, and designated as P. coarctata serine-rich-protein (PcSrp) encoding gene. Northern analysis and in situ mRNA hybridization revealed the expression of PcSrp in the salt stressed roots and rhizome of P. coarctata. However, no such expression was seen in the salt stressed leaves and in the unstressed tissues of root, rhizome and leaf, indicating that PcSrp is under the control of a salt-inducible tissue-specific promoter. In yeast, the PcSrp conferred increased NaCl tolerance, implicating its role in salinity tolerance at cellular level. Further, PcSrp was cloned downstream to rice Actin-1 promoter and introduced into finger millet through particle-inflow-gun method. Transgenic plants expressing PcSrp were able to grow to maturity and set seed under 250 mM NaCl stress. The untransformed control plants by contrast failed to survive under similar salt stress. The stressed roots of transgenic plants invariably accumulated higher Na⁺ and K⁺ ion contents compared to roots of untransformed plants; whereas, shoots of transgenics accumulated lower levels of both the ions than that of untransformed plants under identical stress, clearly suggesting the involvement of PcSrp in ion homeostasis contributing to salt tolerance.     

The expression of a barley HvNAS1 nicotianamine synthase gene promoter-gus fusion gene in transgenic tobacco is induced by Fe-deficiency in roots

Nicotianamine (NA) is a precursor for mugineic acid-family phytosiderophores, which are a critical component of the Fe aquisition process in graminaceous plants. In addition, nicotianamine synthase (NAS) is strongly induced in these plants by Fe deficiency. NA is essential for Fe metabolism also in dicots, but NAS is not induced by Fe deficiency. We introduced a barley HvNAS1 promoter-gus fusion gene into tobacco. GUS activity was induced in the roots of these plants by Fe deficiency, and was constitutively expressed at a low level in their leaves.     

The role of nicotianamine synthase in response to Fe nutrition status in Gramineae

Nicotianamine is an intermediate for the biosynthesis of mugineic acid-family phytosiderophores (MAs) in the Gramineae and a key substance for iron metabolism in dicots. Nicotianamine synthase catalyzes the formation of nicotianamine from S-adenosylmethionine. Nicotianamine synthase activity was induced in barley roots at the 3rd day after withholding Fe supply and declined within one day followmg the supply of Fe³⁺-epihydroxymugineic acid. The induction of nicotianamine synthase activity by Fe-deficiency was observed also in sorghum, maize, and rye, and the level of nicotianamine synthase activity was highly associated with the MAs secreted among graminaceous plant tested. Therefore, the nicotianamine synthase gene may be a suitable candidate for making a transgenic plant tolerant to Fe-deficiency.Abbreviations p-APMSF (p-amidinophenyl) methanesulfonylfluoride hydrochloride - NA nicotianamine - DMA 2-deoxymugineic acid - E-64 trans-epoxysuccinyl-leucylamido-(4-guanidino) butane - epiHMA 3-epihydroxymugineic acid - MAs mugineic acid-family phytosiderophores which include deoxymugineic acid, mugineic acid, hydroxymugineic acid, epihydroxymugineic acid and avenic acid - PVP polyvinylpyrrolidone - SAM S-adenosylmethionine