Deoxymugineic acid increases Zn translocation in Zn-deficient rice plants

Deoxymugineic acid (DMA) is a member of the mugineic acid family phytosiderophores (MAs), which are natural metal chelators produced by graminaceous plants. Rice secretes DMA in response to Fe deficiency to take up Fe in the form of Fe(III)–MAs complex. In contrast with barley, the roots of which secrete MAs in response to Zn deficiency, the amount of DMA secreted by rice roots was slightly decreased under conditions of low Zn supply. There was a concomitant increase in endogenous DMA in rice shoots, suggesting that DMA plays a role in the translocation of Zn within Zn-deficient rice plants. The expression of OsNAS1 and OsNAS2 was not increased in Zn-deficient roots but that of OsNAS3 was increased in Zn-deficient roots and shoots. The expression of OsNAAT1 was also increased in Zn-deficient roots and dramatically increased in shoots; correspondingly, HPLC analysis was unable to detect nicotianamine in Zn-deficient shoots. The expression of OsDMAS1 was increased in Zn-deficient shoots. Analyses using the positron-emitting tracer imaging system (PETIS) showed that Zn-deficient rice roots absorbed less ⁶²Zn-DMA than ⁶²Zn²⁺. Importantly, supply of ⁶²Zn-DMA rather than ⁶²Zn²⁺ increased the translocation of ⁶²Zn into the leaves of Zn-deficient plants. This was especially evident in the discrimination center (DC). These results suggest that DMA in Zn-deficient rice plants has an important role in the distribution of Zn within the plant rather than in the absorption of Zn from the soil. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Motofumi Suzuki and Takashi Tsukamoto equally contributed to this work.     

Induced activity of adenine phosphoribosyltransferase (APRT) in iron-deficiency barley roots: a possible role for phytosiderophore production

To isolate the genes involved in the response of graminaceous plants to Fe-deficient stress, a protein induced by Fe-deficiency treatment was isolated from barley (Hordeum vulgare L.) roots. Based on the partial amino acid sequence of this protein, a cDNA (HvAPT1) encoding adenine phosphoribosyltransferase (APRT: EC 2.4.2.7) was cloned from a cDNA library prepared from Fe-deficient barley roots. Southern analysis suggested that there were at least two genes encoding APRT in barley. Fe deficiency increased HvAPT1 expression in barley roots and resupplying Fe to the Fe-deficient plants rapidly negated the increase in HvAPT1 mRNA. Analysis of localization of HvAPT1-sGFP fusion proteins in tobacco BY-2 cells indicated that the protein from HvAPT1 was localized in the cytoplasm of cells. Consistent with the results of Northern analysis, the enzymatic activity of APRT in barley roots was remarkably increased by Fe deficiency. This induction of APRT activity by Fe deficiency was also observed in roots of other graminaceous plants such as rye, maize, and rice. In contrast, the induction was not observed to occur in the roots of a non-graminaceous plant, tobacco. Graminaceous plants generally synthesize the mugineic acid family phytosiderophores (MAs) in roots under Fe-deficient conditions. In this paper, a possible role of HvAPT1 in the biosynthesis of MAs related to adenine salvage in the methionine cycle is discussed.     

Immunological characterization of a 36 kDa Fe deficiency specific peptide in barley roots

In a previous paper we reported that an acidic 36 kDa peptide is the most strongly induced peptide among several peptides induced by Fe deficiency in barley roots. In this paper, polyclonal antibodies were raised against the 36 kDa peptide. This peptide appeared in the roots of all the graminaceous species tested (barley, rye, wheat, oat, maize, sorghum and rice) in response to Fe deficiency. More of the peptide was found in the roots of graminaceous species which secrete higher amounts of mugineic acids (MAs) under Fe deficient nutrition status. Induction of the 36 kDa peptide was first observed on the third day of Fe deficiency, rising to a maximum value on the seventh day. The trend has a positive correlation with secretion of MAs during Fe deficiency. Further, resupply of Fe resulted in a decrease in peptide production on the second day, reaching a control level on the seventh day. The rate of decrease in peptide production was observed to be slower than that of MA secretion. Other nutrient stresses such as B excess, B deficiency, Cu excess, Cu deficiency, Mn excess, Mn deficiency, Zn excess and Zn deficiency induced far less of the peptide. The specific expression of the 36 kDa peptide in roots of graminaceous species under Fe deficiency suggested the positive association of the peptide with a specific Fe deficiency tolerance mechanism in graminaceous plants.     

Cloning two genes for nicotianamine aminotransferase, a critical enzyme in iron acquisition (Strategy II) in graminaceous plants

Nicotianamine aminotransferase (NAAT), the key enzyme involved in the biosynthesis of mugineic acid family phytosiderophores (MAs), catalyzes the amino transfer of nicotianamine (NA). MAs are found only in graminaceous plants, although NA has been detected in every plant so far investigated. Therefore, this amino transfer reaction is the first step in the unique biosynthesis of MAs that has evolved in graminaceous plants. NAAT activity is dramatically induced by Fe deficiency and suppressed by Fe resupply. Based on the protein sequence of NAAT purified from Fe-deficient barley (Hordeum vulgare) roots, two distinct cDNA clones encoding NAAT, naat-A and naat-B, were identified. Their deduced amino acid sequences were homologous to several aminotransferases, and shared consensus sequences for the pyridoxal phosphate-binding site lysine residue and its surrounding residues. The expression of both naat-A and naat-B is increased in Fe-deficient barley roots, while naat-B has a low level of constitutive expression in Fe-sufficient barley roots. No detectable mRNA from either naat-A or naat-B was present in the leaves of either Fe-deficient or Fe-sufficient barley. One genomic clone with a tandem array of naat-B and naat-A in this order was identified. naat-B and naat-A each have six introns at the same locations. The isolation of NAAT genes will pave the way to understanding the mechanism of the response to Fe in graminaceous plants, and may lead to the development of cultivars tolerant to Fe deficiency that can grow in calcareous soils.     

Nicotianamine synthase gene expression differs in barley and rice under Fe-deficient conditions

Nicotianamine (NA) is an intermediate in the biosynthetic pathway of the mugineic acid family phytosiderophores (MAs), which are crucial components of the iron acquisition apparatus of graminaceous plants. In non-graminaceous plants, NA is thought to be an essential chelator for metal cation homeostasis. Thus NA plays a key role in Fe metabolism and homeostasis in all higher plants. Nicotianamine synthase (NAS, EC 2.5.1.43) catalyzes the trimerization of S-adenosylmethionine to form one molecule of NA. Barley, a plant that is resistant to Fe deficiency, secretes large amounts of MAs, whereas rice, a plant that is susceptible to Fe deficiency, secretes only small amounts. In this study we isolated a genomic fragment containing HvNAS1 from barley and three rice cDNA clones, osnas1, osnas2 and osnas3, from Fe-deficient rice roots. We also isolated a genomic fragment containing both OsNAS1 and OsNAS2. In contrast to barley, in which Fe deficiency induces the expression of NAS genes only in roots, Fe deficiency in rice induced NAS gene expression in both roots and chlorotic leaves. The amounts of endogenous NA in both the roots and leaves were higher than in barley. We introduced barley genomic DNA fragments containing HvNAS1 with either 9 or 2 kb of the 5'-flanking region into rice, using Agrobacterium-mediated transformation. Fe deficiency induced HvNAS1 expression in both roots and leaves of the transgenic rice, as occurs with rice NAS genes. Barley and rice NAS genes are compared in a discussion of alteration of the NAS genes during adaptation to Fe deficiency.     

Differential regulation of cadmium-inducible expression of iron-deficiency-responsive genes in tobacco and barley

The expression of four representative iron-deficiency-responsive genes from tobacco ( NtIRT1 and NtYSL1 ) and barley ( HvIDS2 and HvYS1 ) plants were evaluated in each host plant in response to iron deficiency (ΔFe), cadmium exposure (+Cd) or both (ΔFe + Cd). These conditions significantly enhanced NtIRT1 and HvIDS2 expression in roots, whereas NtYSL1 and HvYS1 expression was similar in shoots and roots. NtIRT1 expression under +Cd and ΔFe + Cd was lower than that under ΔFe, whereas the expression of NtYSL1 , HvIDS2 and HvYS1 in roots under +Cd and ΔFe + Cd was similar or higher than that under ΔFe. A time-course experiment showed that NtIRT1 expression under +Cd and ΔFe was regulated similarly throughout the experiment [expressed between 3 and 21 days after treatment (DAT)]. NtYSL1 expression under +Cd and ΔFe began at 1 DAT; expression soon disappeared under ΔFe, whereas it continued to 21 DAT under +Cd. The timing of HvIDS2 and HvYS1 expression under +Cd (between 1 and 5 DAT) was earlier than that under ΔFe (between 5 and 21 DAT). Notably, no Fe deficit occurred in any parts of these plants when grown under +Cd, except for tobacco shoots, even when the genes were highly expressed. Thus, some expression under +Cd differed from that under ΔFe. It is possible that both the genuine Fe-deficiency-responsive mechanism and an unidentified mechanism, which can be directly regulated by Cd, contribute to gene expression to maintain metal homeostasis within the plant.     

Response of barley plants to Fe deficiency and Cd contamination as affected by S starvation

Both Fe deficiency and Cd exposure induce rapid changes in the S nutritional requirement of plants. The aim of this work was to characterize the strategies adopted by plants to cope with both Fe deficiency (release of phytosiderophores) and Cd contamination [production of glutathione (GSH) and phytochelatins] when grown under conditions of limited S supply. Experiments were performed in hydroponics, using barley plants grown under S sufficiency (1.2 mM sulphate) and S deficiency (0 mM sulphate), with or without Fe(III)-EDTA at 0.08 mM for 11 d and subsequently exposed to 0.05 mM Cd for 24 h or 72 h. In S-sufficient plants, Fe deficiency enhanced both root and shoot Cd concentrations and increased GSH and phytochelatin levels. In S-deficient plants, Fe starvation caused a slight increase in Cd concentration, but this change was accompanied neither by an increase in GSH nor by an accumulation of phytochelatins. Release of phytosiderophores, only detectable in Fe-deficient plants, was strongly decreased by S deficiency and further reduced after Cd treatment. In roots Cd exposure increased the expression of the high affinity sulphate transporter gene (HvST1) regardless of the S supply, and the expression of the Fe deficiency-responsive genes, HvYS1 and HvIDS2, irrespective of Fe supply. In conclusion, adequate S availability is necessary to cope with Fe deficiency and Cd toxicity in barley plants. Moreover, it appears that in Fe-deficient plants grown in the presence of Cd with limited S supply, sulphur may be preferentially employed in the pathway for biosynthesis of phytosiderophores, rather than for phytochelatin production.     

Alfalfa genotypes differ in their ability to tolerate zinc deficiency

Response of 13 alfalfa (Medicago sativa L.) genotypes to varied Zn supply (+Zn: 2 mg kg⁻¹ soil, −Zn: no added Zn) was studied in a pot experiment under controlled environmental conditions. Plants were grown for four weeks in a Zn-deficient siliceous sandy soil. Plants grown at no added Zn showed typical Zn deficiency symptoms i.e. interveinal chlorosis of leaves, yellowish-white necrotic lesions on leaf blades, necrosis of leaf margins, smaller leaves and a marked reduction in growth. There was solute leakage from the leaves of Zn-deficient plants, while no solute leakage from Zn-sufficient plants. The ratios of P:Zn, Fe:Zn, Cu:Zn and Mn:Zn in Zn-deficient plants were extremely high compared with Zn-sufficient plants indicating disturbance of P:Zn, Fe:Zn, Cu:Zn and Mn:Zn balance within plant system by Zn deficiency. Genotypes differed markedly in Zn efficiency based on shoot dry matter production. Alfalfa genotypes also differed markedly in P:Zn ratio, Cu:Zn ratio and Fe:Zn ratio under —Zn treatment. The shoot dry weight, shoot:root ratio, chlorophyll content of fresh leaf tissue, solute leakage from the leaves, Zn uptake and distribution of Zn in shoots and roots were the most sensitive parameters of Zn efficiency. Zn-efficient genotypes had less solute leakage but higher shoot:root ratio and higher Zn uptake compared with Zn-inefficient genotypes. Under —Zn treatment, Zn-inefficient genotypes had less Zn partitioning to shoots (33–37%) and more Zn retained in roots (63–67%), while Zn-efficient genotypes had about equal proportions of Zn in roots (50%) and shoots (50%). This revised version was published online in June 2006 with corrections to the Cover Date.     

Root exudation and Fe uptake and transport in wheat genotypes differing in tolerance to Zn deficiency

Tolerance to Zn deficiency in wheat germplasm may be inversely related to uptake and transport of Fe to shoots. The present study examined eight bread (Triticum aestivum) and two durum (T. turgidum L. conv. durum) wheat genotypes for their capacity to take up and transport Fe when grown under either Fe or Zn deficiency. Bread wheat genotypes Aroona, Excalibur and Stilleto showed tolerance to Zn and Fe deficiency, while durum wheat genotypes are clearly less tolerant to either deficiency. Roots of bread wheats tolerant to Zn deficiency exuded more phytosiderophores than sensitive bread and durum genotypes. Greater amounts of phytosideophores were exuded by roots grown under Fe than Zn deficiency. A relatively poor relationship existed between phytosiderophore exudation or the Fe uptake rate and relative shoot growth under Fe deficiency. At advanced stages of Zn deficiency, genotypes tolerant to Zn deficiency (Aroona and Stilleto) had a greater rate of Fe uptake than other genotypes. Zinc deficiency depressed the rate of Fe transport to shoots in all genotypes in early stages, while advanced Zn deficiency had the opposite effect. Compared with Zn-sufficient plants, 17-day-old Zn-deficient plants of genotypes tolerant to Zn deficiency had a lower rate of Fe transport to shoots, while genotypes sensitive to Zn deficiency (Durati, Yallaroi) had the Fe transport rate increased by Zn deficiency. A proportion of total amount of Fe taken up that was transported to shoots increased with duration of either Fe or Zn deficiency. It is concluded that greater tolerance to Zn deficiency among wheat genotypes is associated with the increased exudation of phytosiderophores, an increased Fe uptake rate and decreased transport of Fe to shoots. This revised version was published online in June 2006 with corrections to the Cover Date.     

Expression of iron-acquisition-related genes in iron-deficient rice is co-ordinately induced by partially conserved iron-deficiency-responsive elements

Rice plants (Oryza sativa L.) utilize the iron chelators known as mugineic acid family phytosiderophores (MAs) to acquire iron from the rhizosphere. Synthesis of MAs and uptake of MA-chelated iron are strongly induced under conditions of iron deficiency. Microarray analysis was used to characterize the expression profile of rice in response to iron deficiency at the genomic level. mRNA extracted from iron-deficient or iron-sufficient rice roots or leaves was hybridized to a rice array containing 8987 cDNA clones. An induction ratio of greater than 2.0 in roots was observed for 57 genes, many of which are involved in iron-uptake mechanisms, including every identified or predicted step in the methionine cycle and the biosynthesis of MAs from methionine. Northern analysis confirmed that the expression of genes encoding every step in the methionine cycle is thoroughly induced by iron deficiency in roots, and almost thoroughly induced in leaves. A promoter search revealed that the iron-deficiency-induced genes related to iron uptake possessed sequences homologous to the iron-deficiency-responsive cis-acting elements IDE1 and IDE2 in their promoter regions, at a higher rate than that showing no induction under Fe deficiency. These results suggest that rice genes involved in iron acquisition are co-ordinately regulated by conserved mechanisms in response to iron deficiency, in which IDE-mediated regulation plays a significant role.     

Identification and localisation of the rice nicotianamine aminotransferase gene OsNAAT1 expression suggests the site of phytosiderophore synthesis in rice

Rice plants (Oryza sativa L.) take up iron using iron-chelating compounds known as mugineic acid family phytosiderophores (MAs). In the biosynthetic pathway of MAs, nicotianamine aminotransferase (NAAT) catalyses the key step from nicotianamine to the 3′′-keto form. In the present study, we identified six rice NAAT genes (OsNAAT1–6) by screening a cDNA library made from Fe-deficient rice roots and by searching databases. Among the NAAT homologues, OsNAAT1 belongs to a subgroup containing barley functional NAAT (HvNAAT-A and HvNAAT-B) as well as a maize homologue cloned by cDNA library screening (ZmNAAT1). Northern blot and RT-PCR analysis showed that OsNAAT1, but not OsNAAT2–6, was strongly up-regulated by Fe deficiency, both in roots and shoots. The OsNAAT1 protein had NAAT enzyme activity in vitro, confirming that the OsNAAT1 gene encodes functional NAAT. Promoter–GUS analysis revealed that OsNAAT1 was expressed in companion and pericycle cells adjacent to the protoxylem of Fe-sufficient roots. In addition, expression was induced in all cells of Fe-deficient roots, with particularly strong GUS activity evident in the companion and pericycle cells. OsNAAT1 expression was also observed in the companion cells of Fe-sufficient shoots, and was clearly induced in all the cells of Fe-deficient leaves. These expression patterns highly resemble those of OsNAS1, OsNAS2 and OsDMAS1, the genes responsible for MAs biosynthesis for Fe acquisition. These findings strongly suggest that rice synthesises MAs in whole Fe-deficient roots to acquire Fe from the rhizosphere, and also in phloem cells to maintain metal homeostasis facilitated by MAs-mediated long-distance transport.     

Overcoming Fe deficiency by a transgenic approach in rice

Iron (Fe) is an essential microelement for plant growth. Fe availability is particularly limited on calcareous soils, which have high pH. Approximately 30% of the world's soils are considered calcareous with low Fe availability, which results in extensive areas of Fe deficiency in plants. Some graminaceous plants are known to secrete high amounts of mugineic acid family phytosiderophores (MAs) under Fe deficiency. This Fe acquisition system is called the Strategy-II mechanism. Tolerance to Fe deficiency in graminaceous plants is thought to depend on the quantity of MAs secreted by plants under Fe deficiency stress. This system was utilized to enhance the tolerance of rice to low Fe availability. Transgenic rice expressing the barley naat genes, one of the genes for the enzymes on the biosynthetic pathway of MAs, showed tolerance to low Fe availability when grown in a calcareous soil.     

Zinc deficiency-induced phytosiderophore release by the Triticaceae is not consistently expressed in solution culture

The effects of zinc (Zn) and iron (Fe) deficiencies on phytosiderophore (PS) exudation by three barley (Hordeum vulgare L.) cultivars differing in Zn efficiency were assessed using chelator-buffered nutrient solutions. A similar study was carried out with four wheat (Triticum aestivum L. and T. durum Desf.) cultivars, including the Zn-efficient Aroona and Zn-inefficient Durati. Despite severe Zn deficiency, none of the barley or wheat cultivars studied exhibited significantly elevated PS release rates, although there was significantly enhanced PS exudation under Fe deficiency. Aroona and Durati wheats were grown in a further study of the effects of phosphate (P) supply on PS release, using 100 μM KH₂PO₄ as high P, or solid hydroxyapatite as a supply of low-release P. Phytosiderophore exudation was not increased due to P treatment under control or Zn-deficient conditions, but was increased by Fe deficiency. Accumulation of P in shoots of Zn- and Fe-deficient plants was seen in both P treatments, somewhat more so under the KH₂PO₄ treatment. Zinc-efficient wheats and grasses have been previously shown to exude more PS under Zn deficiency than Zn-inefficient genotypes. We did not observe Zn-deficiency-induced PS release and were unable to replicate the results of previous researchers. The tendency for Zn deficiency to induce PS release seems to be method dependent, and we suggest that all reported instances may be explained by an induced physiological deficiency of Fe. Received: 25 October 1999 / Accepted: 3 December 1999     

Ecotypes of Holcus lanatus Tolerant to Zinc Toxicity also Tolerate Zinc Deficiency

Genotypes tolerant to zinc (Zn) toxicity, if they accumulateZn in their roots, may grow better than Zn-sensitive genotypes,even in Zn-deficient soil. In the present study, Holcus lanatusL. ecotypes differing in tolerance to Zn toxicity were grownin Zn-deficient Laffer soil which was amended with Zn to createa range of conditions from Zn deficiency to Zn toxicity. IncreasingZn additions to the soil, up to the sufficiency level, improvedgrowth of all ecotypes. At toxic levels of added Zn, the Zn-sensitiveecotype suffered a greater decrease in growth than the Zn-tolerantecotypes. All ecotypes accumulated more Zn in roots than inshoots, with root concentrations exceeding 8 g Zn kg^-1dry weightin extreme cases. When grown in Zn-deficient or Zn-sufficientsoil (up to 0.5 mg Zn kg^-1soil added), ecotypes tolerant toZn toxicity took up more Zn, grew better and had greater rootand shoot Zn concentration than the control (Zn-sensitive ecotype).Zn-tolerant ecotypes transported more Zn, copper (Cu) and iron(Fe) from roots to shoots in comparison with the Zn-sensitiveecotype. The average Zn uptake rate from Zn-deficient soil (noZn added) was greater in the Zn-tolerant ecotypes than in theZn-sensitive ecotype. In conclusion, ecotypes of H. lanatusthat are tolerant to Zn toxicity also tolerate Zn deficiencybetter than the Zn-sensitive ecotype because of their greatercapacity for taking up Zn from Zn-deficient soil. This is thefirst report of the coexistence of traits for tolerance to Zntoxicity and Zn deficiency in a single plant genotype. Copyright2000 Annals of Botany Company Copper, heavy metal, Holcus lanatus, iron, zinc deficiency, zinc toxicity     

Mechanism of phosphorus-induced zinc deficiency in cotton. I. Zinc deficiency-enhanced uptake rate of phosphorus

The effect of withholding Zn on the uptake, translocation and accumulation of P was studied in cotton plants ( Gossypium hirsutum L. cv. Deltapine 15/21) grown in nutrient solutions under controlled environmental conditions. The influence of P on the uptake rate, translocation and distribution of ⁶⁵Zn in the plants was also examined. Increasing the P supply resulted in severe Zn deficiency symptoms (interveinal chlorosis) as well as P toxicity symptoms, which were characterized by leaf puckering and grayish-brown marginal necrosis. Zinc deficiency markedly increased the uptake and translocation rates of P over the whole concentration range tested (5x10^-5 to 1.25x10^-3 M ). Uptake and translocation rates of P increased with both level of P and severity of Zn deficiency. This often caused P toxicity symptoms on Zn-deficient leaves. In contrast to P, the concentrations of K and Mg in the leaves were not affected by Zn deficiency. Similar results were obtained for sunflower ( Helianthus annuus L.) and buckwheat ( Fagopyrum esculentum Moench) plants. Higher P concentrations in Zn-deficient leaves or shoots could not be attributed wholly to reduced shoot growth. This was also evident when Zn deficiency was compared with other micronutrient (Fe, Mn, and Cu) deficiencies. Only Zn-deficient plants showed enhanced uptake and translocation of P. In experiments with ⁶⁵Zn, a high P supply did not depress uptake and translocation of Zn. From the results obtained it is concluded that the P-induced Zn deficiency in cotton, as well as in other species, is primarily caused by enhanced P uptake and translocation and not by inhibition of Zn uptake.     

Growth and Nutrient Uptake by Barley (Hordeum vulgare L. cv Herta): Studies Using an N-(2-Hydroxyethyl)ethylenedinitrilotriacetic Acid-Buffered Nutrient Solution Technique (I. Zinc Ion Requirements)

The critical range of Zn2+ activity in nutrient solution required for optimum growth of barley (Hordeum vulgare L. cv Herta) was studied using the synthetic chelating agent N-(2-hydroxyethyl)ethylenedinitrilotriacetic acid to buffer micronutrient metal ions. The activity of Zn2+ was varied over a wide range from approximately 0.1 x 10-11 to 22 x 10-11 M Zn2+. The dry weight of barley shoots reached a maximum at Zn2+ activities above approximately 3 x 10-11 M and was clearly depressed when Zn2+ activities were below about 1 x 10-11 M. The relationship in shoots between dry weight and Zn concentrations supports the view that there is a critical Zn concentration of about 25 [mu]g g-1 dry weight in whole shoots of barley seedlings. When Zn2+ activities in solution were near or below approximately 3 x 10-11 M, barley shoots accumulated higher concentrations of P, Mn, Ca, Mg, and Na, whereas Cu concentrations were reduced. P and Mn began to accumulate in the shoots before differences in dry weights were apparent and provided the earliest index of Zn deficiency. In Zn-deficient roots, concentrations of Ca and Mg increased by 25 to 30%, and those of Fe and Mn more than doubled. Zn appears to play a special role in regulating uptake of several mineral nutrients in barley.