Genes controlling hydroxylations of phytosiderophores are located on different chromosomes in barley (Hordeum vulgare L.)

Phytosiderophores, mugineic acids, have been demonstrated to be involved in Fe acquisition in gramineous plants. In this study, chromosomal arm locations of genes encoding for biosynthesis of various phytosiderophores were identified in a cultivar of barley (Hordeum vulgare L. cv. Betzes). Using wheat (Triticum aestivum L. cv. Chinese Spring)-barley (cv. Betzes) ditelosomic addition lines for 4HS and 4HL, a gene for hydroxylation of 2′-deoxymugineic acid to mugineic acid was localized to the long arm of barley chromosome 4H. To locate the gene for hydroxylation of mugineic acid to 3-epihydroxymugineic acid, hybrids between the 4H addition line and other wheat-barley addition lines were studied. Only a hybrid between 4H and 7H addition lines produced 3-epihydroxymugineic acid. The gene was further localized to the long arm of chromosome 7H by feeding mugineic acid to ditelosomic addition lines for 7HS and 7HL. A new phytosiderophore was discovered in both 7H and 7HL addition lines, which was identified to be 3-epihydroxy-2′-deoxymugineic acid by detailed nuclear magnetic resonance studies. These results revealed that in barley there are two pathways from 2′-deoxymugineic acid to 3-epihydroxymugineic acid: 2′-deoxymugineic acid → mugineic acid → 3-epihydroxymugineic acid and 2′-deoxymugineic acid → 3-epihydroxy-2′-deoxymugineic acid → 3-epihydroxymugineic acid. Barley genes encoding for the hydroxylations of phytosiderophores are located in different chromosomes and each gene hydroxylates different C-positions: the long arm of chromosome 4H carries the gene for hydroxylating the C-2′ position and the long arm of chromosome 7H carries the gene for hydroxylating the C-3 position of the azetidine ring. Received: 10 August 1998 / Accepted: 30 September 1998     

Nicotianamine aminotransferase activities are correlated to the phytosiderophore secretions under Fe-deficient conditions in Gramineae

The activities of nicotianamine aminotransferase, one of theenzymes for the biosynthesis of mugineic acid-family phytosiderophores,were examined in six graminaceous species. The enzyme activitieswere induced by Fe-deficiency treatments in all species testedand had a considerable correlation to the amounts of secretedmugineic acids. Key words: Fe-deficiency, mugineic acid, nicotianamine aminotransferase, phytosiderophore, Gramineae     

Hydroxylated phytosiderophore species possess an enhanced chelate stability and affinity for iron(III)

Graminaceous plant species acquire soil iron by the release of phytosiderophores and subsequent uptake of iron(III)-phytosiderophore complexes. As plant species differ in their ability for phytosiderophore hydroxylation prior to release, an electrophoretic method was set up to determine whether hydroxylation affects the net charge of iron(III)-phytosiderophore complexes, and thus chelate stability. At pH 7.0, non-hydroxylated (deoxymugineic acid) and hydroxylated (mugineic acid; epi-hydroxymugineic acid) phytosiderophores form single negatively charged iron(III) complexes, in contrast to iron(III)-nicotianamine. As the degree of phytosiderophore hydroxylation increases, the corresponding iron(III) complex was found to be less readily protonated. Measured pKa values of the amino groups and calculated free iron(III) concentrations in presence of a 10-fold chelator excess were also found to decrease with increasing degree of hydroxylation, confirming that phytosiderophore hydroxylation protects against acid-induced protonation of the iron(III)-phytosiderophore complex. These effects are almost certainly associated with intramolecular hydrogen bonding between the hydroxyl and amino functions. We conclude that introduction of hydroxyl groups into the phytosiderophore skeleton increases iron(III)-chelate stability in acid environments such as those found in the rhizosphere or the root apoplasm and may contribute to an enhanced iron acquisition.     

The binding of aluminum to mugineic acid and related compounds as studied by potentiometric titration

The phytosiderophores, mugineic acid (MA) and epi-hydroxymugineic acid (HMA), together with a related compound, nicotianamine (NA), were investigated for their ability to bind Al(III). Potentiometric titration analysis demonstrated that MA and HMA bind Al(III), in contrast to NA which does not under normal physiological conditions. With MA and HMA, in addition to the Al complex (AlL), the protonated (AlLH) and deprotonated (AlLH₋₁) complexes were identified from an analysis of titration curves, where L denotes the phytosiderophore form in which all the carboxylate functions are ionized. The equilibrium formation constants of the Al(III) phytosiderophore complexes are much smaller than those of the corresponding Fe(III) complexes. The higher selectivity of phytosiderophores for Fe(III) over Al(III) facilitates Fe(III) acquisition in alkaline conditions where free Al(III) levels are higher than free Fe(III) levels.     

Detection of two distinct isozymes of nicotianamine aminotransferase in Fe-deficient barley roots

Two isozymes of nicotianamine aminotransferase, involved inthe biosynthesis of phytosiderophores, were detected in Fe-deficientbarley roots. They showed different molecular masses and kineticparameters. Furthermore, one of them was strongly induced byan Fe-deficiency treatment, whereas the other was not. Key words: Fe-deficiency, Amugineic acid, nicotianamine aminotransferase, phytosiderophore, barley roots     

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     

Spontaneous chromosomal rearrangements in cultivated and wild barleys

Summary Four of 1,240 cultivated barley lines collected from different regions of the world and 3 of 120 lines of wild barley, Hordeum spontaneum C. Koch, carry spontaneous reciprocal translocations. Break-point positions and rearrangements in the interchanged chromosomes have been examined by both test crosses and Giemsa banding techniques. The four translocation lines in cultivated barley were all of Ethiopian origin and have the same translocation involving chromosomes 2 and 4. The breakpoints are at the centromeres of both chromosomes, resulting in interchanged chromosomes 2S+4S and 2L+4L (S=short arm, L=long arm). A wild barley line, Spont.II, also has translocated chromosomes 2 and 4 which are broken at the centromeres. The resultant chromosomes are, however, 2S+4L and 2L+4S. Another wild barley line, Spont.S-4, has interchanged chromosomes with breakpoints in the short arm of chromosome 3 and the long arm of chromosome 7. In addition, this line has a paracentric inversion in the short arm of chromosome 7 that includes a part of nucleolar constriction, resulting in two tandemly arranged nucleolar constrictions. The third wild barley line, Spont.S-7, has interchanged chromosomes with breakpoints in the long arms of both chromosomes 3 and 6. The translocated chromosome 3 is metacentric and the translocated chromosome 6 has a long arm similar in length to the long arm of chromosome 7.     

Why are young rice plants highly susceptible to iron deficiency?

The reason why young rice plant is highly susceptible to Fe-deficiency was clarified as follows: Among Gramineae plants rice secreted a very low amount of deoxy-MA as a phytosiderophore even under Fe-deficiency, and the secretion by rice ceased within 10 days under Fe-deficiency although barley secreted MAs during a period of more than one month. When iron depletion continued, the rice root tips become chimeric and epidermal cells became necrotic. The mitochondrial membrane systems in the cortex cells were also severely damaged. Iron starvation occurred even in the mitochondria, and energy charge in the root decreased. This reduced energy charge has firstly diminished the secretion activity of deoxy-MA from the roots, secondly reduced the activity of the transporter which absorb deoxy-MA-Fe^III chelate and finally reduced the synthesis of deoxy-MA from metionine. Consequently, the depletion of Fe^II in the shoot was induced and severe chlorosis rapidly developed in the young rice plant under Fe-deficiency.Abbreviations DCCD dicyclohexylcarbodiimide - CCCP carbonylcyanide-m-chlorophenylhydrazone - MA mugineic acid - MAs mugineic acid-family phytosiderophores, it contains deoxy-MA, MA, epihydroxy-MA, hydroxy-MA, avenic acid and distichonic acid     

Incorporation of 15N and 14C of methionine into the mugineic acid family of phytosiderophores in iron-deficient barley roots

The role of methionine as a precursor in mugineic acid (MA) biosynthesis was studied by feeding ¹⁵N-ammonium sulfate, ¹⁴C-amino acids, and [1-¹⁴C, ¹⁵N]-methionine to iron-deficient barley roots ( Hordeum vulgare L. cv. Minorimugi), grown hydroponically. The incorporation of isotopes into amino acids was also examined. Methionine appears to be the most efficient precursor of the mugineic acid family (MAs) of phytosiderophores; homoserine was also incorporated into the MAs, but other amino acids such as glutamate, alanine, and γ-amino butyric acid did not act as precursors of MAs. Carbon-14 and ¹⁵N of methionine were incorporated into MAs. This specific incorporation of ¹⁴C and ¹⁵N indicated that the nitrogen atoms of MAs were derived from two molecules of methionine. It is suggested that deoxymugineic acid (DMA) is probably the first phytosiderophore to be synthesized on the biosynthetic pathway of MAs.     

Phytosiderophore release from nodal,primary, and complete root systems in maize

Some maize (Zea mays L.) hybrids grown in high pH soil in Nebraska suffer from severely reduced yields caused by iron (Fe) deficiency chlorosis. Hybrids which recover from early season Fe-deficiency chlorosis and yield well are termed Fe-efficient or tolerant. Most Fe-efficient gramineous species respond to Fe-deficiency stress by releasing phytosiderophores (mugineic acid and its derivatives) into the rhizosphere, thereby increasing Fe availability and uptake of the Fe³⁺-phytosiderophore complex via a high affinity uptake system. Field-grown Fe-efficient maize recovers from Fe-deficiency chlorosis at a stage when nodal roots have become the dominant root system. Quantifying phytosiderophore release from hydroponically grown plants has been proposed as a viable alternative to time-consuming and variable field trials and has been used successfully to delineate among Fe-efficient and Fe-inefficient lines of oat (Avena sativa L.) and wheat (Triticum aestivum L.). Our objectives were (1) to determine if phytosiderophore release differed between nodal- and primary-root systems of maize, and (2) to compare phytosiderophore release from 12 hybrids. Root exudates secreted during daily 4-h collections were analyzed for their Fe-solubilizing ability, which was equated to phytosiderophore release. Nodal root systems released significantly more phytosiderophore than primary- or complete-root systems. In early experiments, an Fe-efficient hybrid (P3279) released more phytosiderophore from nodal roots than an Fe-inefficient hybrid (P3489). Tests of an additional 10 hybrids showed that phytosiderophore release varied significantly among the cultivars but did not clearly distinguish between hybrids classified as Fe-efficient or Fe-inefficient in individual company trials. We recommend using nodal roots when studying Fe-stress response mechanisms in maize.     

The Phytosiderophore Efflux Transporter TOM2 Is Involved in Metal Transport in Rice

Iron is an essential metal element for all living organisms. Graminaceous plants produce and secrete mugineic acid family phytosiderophores from their roots to acquire iron in the soil. Phytosiderophores chelate and solubilize insoluble iron hydroxide in the soil. Subsequently, plants take up iron-phytosiderophore complexes through specific transporters on the root cell membrane. Phytosiderophores are also thought to be important for the internal transport of various transition metals, including iron. In this study, we analyzed TOM2 and TOM3, rice homologs of transporter of mugineic acid family phytosiderophores 1 (TOM1), a crucial efflux transporter directly involved in phytosiderophore secretion into the soil. Transgenic rice analysis using promoter-β-glucuronidase revealed that TOM2 was expressed in tissues involved in metal translocation, whereas TOM3 was expressed only in restricted parts of the plant. Strong TOM2 expression was observed in developing tissues during seed maturation and germination, whereas TOM3 expression was weak during seed maturation. Transgenic rice in which TOM2 expression was repressed by RNA interference showed growth defects compared with non-transformants and TOM3-repressed rice. Xenopus laevis oocytes expressing TOM2 released ¹⁴C-labeled deoxymugineic acid, the initial phytosiderophore compound in the biosynthetic pathway in rice. In onion epidermal and rice root cells, the TOM2-GFP fusion protein localized to the cell membrane, indicating that the TOM2 protein is a transporter for phytosiderophore efflux to the cell exterior. Our results indicate that TOM2 is involved in the internal transport of deoxymugineic acid, which is required for normal plant growth.     

Effective regulation of iron acquisition in graminaceous plants. The role of mugineic acids as phytosiderophores

Discovery of mugineic acids as phytosiderophores has shown that some graminaceous monocotyledonous plants have a different iron acquisition strategy (strategy II) from dicotyledonous and nongraminaceous monocotyledonous plants (strategy I). The process of iron acquisition by strategy II plants can be divided into four main steps: biosynthesis, secretion, solubilization, and uptake, all of which are effectively regulated by different systems. The biosynthesis of mugineic acids is controlled by an on-off system which is operated under the control of iron demand in the plant. All mugineic acids share the same biosynthetic pathway from L-methionine to 2'-deoxymugineic acid, but the subsequent steps differ among plant species and even cultivars. The biosynthesis of mugineic acids is associated with the methionine recycling pathway. The secretion of mugineic acids shows a distinct diumal rhythm. Mugineic acids solubilize sparingly soluble inorganic iron by chelation and possess a high chelation affinity for iron, but not for other polyvalent ions such as Ca²⁺, Mg²⁺ and Al³⁺. The iron uptake process is regulated by a specific uptake system that transports the mugineic acid-Fe(III) complex as an intact molecule. This system specifically recognizes the mugineic acid-Fe(III) complexes, but not other mugineic acid-metal or synthetic chelator-Fe(III) complexes, suggesting that binding sites with strict recognition for stereostructure of the complex are located on the plasma membrane. All these regulatory systems are considered to represent an efficient strategy to acquire adequate amounts of iron and to avoid factors unfavorable for iron acquisition such as high pH, high concentrations of bicarbonate, Ca^2- and Mg²⁺, microbial degradation, and uptake of other metals that are common in calcareous soils.     

Two Related Biosynthetic Pathways of Mugineic Acids in Gramineous Plants

The biosynthesis of mugineic acids was studied by feeding 2H- or 13C-labeled compounds to water-cultured roots in several gramineous plants. The fate of labeled compounds was monitored by using 2H- and 13C-nuclear magnetic resonance. On investigating the proton changes during biosynthesis by feeding D,L-[3,3,4,4-d4]-methionine (98.6% 2H), 2H-labeled 2[prime]-deoxymugineic, mugineic, and 3-epihydroxymugineic acids were isolated from root washings of wheat (Triticum aestivum L. cv Minori), barley (Hordeum vulgare L. cv Minorimugi), and beer barley (Hordeum vulgare L. cv AM Nijo Tochigi), respectively. The 2H-nuclear magnetic resonance study indicated that 12 deuteriums were incorporated into the labeled 2[prime]-deoxymugineic acid, suggesting that three molecules of L-[3,3,4,4-d4]methionine were combined. In comparison, one of the deuteriums at C-2[prime] position in the mugineic acid, and one each of the deuteriums at C-2[prime] and C-3 positions in the 3-epihydroxymugineic acid, were lost. However, all other deuteriums were incorporated in a manner similar to that of the labeled 2[prime]-deoxymugineic acid. When [1,4[prime],4"-13C3]2[prime]-deoxymugineic acid (20% 13C) was fed to oat roots (Avena sativa L. cv Amuri II), avenic acid A, which was 13C enriched at the corresponding positions, was obtained. These results revealed that L-methionine was the precursor for all these mugineic acids and that cleavage of the azetidine ring or hydroxylation of the 2[prime]-deoxymugineic acid produced two related biosynthetic pathways in different gramineous plant species: L-methionine -> 2[prime]-deoxymugineic acid -> avenic acid A in oat; and L-methionine -> 2[prime]-deoxymugineic acid -> mugineic acid -> 3-epihydroxymugineic acid in barley and beer barley.     

Genetic and molecular analyses of resistance to a variant of Puccinia striiformis in barley

Seedlings of 62 Australian barley cultivars and two exotic barley genotypes were assessed for resistance to a variant of Puccinia striiformis, referred to as “Barley Grass Stripe Rust” (BGYR), first detected in Australia in 1998, which is capable of infecting wild Hordeum species and some genotypes of cultivated barley. Fifty-three out of 62 cultivated barley cultivars tested were resistant to the pathogen. Genetic analyses of seedling resistance to BGYR in six Australian barley cultivars and one Algerian barley landrace indicated that they carried either one or two major resistance genes to the pathogen. A single recessive seedling resistance gene, rpsSa3771, identified in Sahara 3771, was located on the long arm of chromosome 1 (7 H), flanked by the restriction fragment length polymorphism (RFLP) markers Xwg420 and Xcdo347 at genetic distances of 12.8 and 21.9 cM, respectively. Mapping resistance to BGYR at adult plant growth stages using the doubled haploid (DH) population Clipper × Sahara 3771 identified two major quantitative trait loci (QTL), one on the long arm of chromosome 3 (3 H) and the second on the long arm of chromosome 1 (7 H), accounting for 26 % and 18 % of the total phenotypic variation, respectively. The QTL located on chromosome 7HL corresponded to seedling resistance gene rpsSa3771 and the second QTL was concluded to correspond to a single APR gene, designated rpsCl, contributed by cultivar Clipper.     

Development of commercially valuable traits in hexaploid triticale lines with Aegilops introgressions as dependent on the genome composition

Introgressive hybridization is an efficient means to improve the genetic diversity of cultivated cereals, including triticale. To identify the triticale lines with Aegilops introgressions, genotyping was carried out with ten lines obtained by crossing hexaploid triticale with genome-substitution forms of the common wheat cultivar Avrora: Avrolata (AABBUU), Avrodes (AABBSS), and Avrotika (AABBTT). The genome composition of the triticale lines was studied by in situ hybridization, and recombination events involving Aegilops and/or common wheat chromosomes were assumed for nine out of the ten lines. Translocations involving rye chromosomes were not observed. Substitutions for rye chromosomes were detected in two lines resulting from crosses with Avrolata. Genomic in situ hybridization (GISH) with Ae. umbellulata DNA and molecular genetic analysis showed that chromosome 1R was substituted with Ae. umbellulata chromosome 1U in one of the lines and that 2R(2U) substitution took place in the other line. Fluorescence in situ hybridization (FISH) with the Spelt 1 and pSc119.2 probes revealed a translocation from Ae. speltoides to the long arm of chromosome 1B in one of the two lines resulting from crosses with Avrodes and a translocation in the long arm of chromosome 7B in the other line. In addition, the pSc119.2 probe revealed chromosome 1B rearrangements in four lines resulting from crosses with Avrolata and in a line resulting from crosses with Avrotika. The lines were tested for main productivity parameters. A negative effect on all productivity parameters was demonstrated for Ae. umbellulata chromosome 2U. The overwinter survival in all of the lines was similar to or even higher than in the original triticale cultivars. A substantial increase in winter resistance as compared with the parental cultivar was observed for the line carrying the T7BS-7SL translocation. The line with the 1R(1U) chromosome substitution seemed promising for the baking properties of triticale.     

Development of commercially valuable traits in hexaploid triticale lines with <Emphasis Type="Italic">Aegilops</Emphasis> introgressions as dependent on the genome composition

Introgressive hybridization is an efficient means to improve the genetic diversity of cultivated cereals, including triticale. To identify the triticale lines with Aegilops introgressions, genotyping was carried out with ten lines obtained by crossing hexaploid triticale with genome-substitution forms of the common wheat cultivar Aurora: Aurolata (AABBUU), Aurodes (AABBSS), and Aurotika (AABBTT). The genome composition of the triticale lines was studied by in situ hybridization, and recombination events involving Aegilops and/or common wheat chromosomes were assumed for nine out of the ten lines. Translocations involving rye chromosomes were not observed. Substitutions for rye chromosomes were detected in two lines resulting from crosses with Aurolata. Genomic in situ hybridization (GISH) with Ae. umbellulata DNA and molecular genetic analysis showed that chromosome 1R was substituted with Ae. umbellulata chromosome 1U in one of the lines and that 2R(2U) substitution took place in the other line. Fluorescence in situ hybridization (FISH) with the Spelt1 and pSc119.2 probes revealed a translocation from Ae. speltoides to the long arm of chromosome 1B in one of the two lines resulting from crosses with Aurodes and a translocation in the long arm of chromosome 7B in the other line. In addition, the pSc119.2 probe revealed chromosome 1B rearrangements in four lines resulting from crosses with Aurolata and in a line resulting from crosses with Aurotika. The lines were tested for main productivity parameters. A negative effect on all productivity parameters was demonstrated for Ae. umbellulata chromosome 2U. The overwinter survival in all of the lines was similar to or even higher than in the original triticale cultivars. A substantial increase in winter resistance as compared with the parental cultivar was observed for the line carrying the T7BS-7SL translocation. The line with the 1R(1U) chromosome substitution seemed promising for the baking properties of triticale.     

Three nicotianamine synthase genes isolated from maize are differentially regulated by iron nutritional status

Nicotianamine synthase (NAS) is an enzyme that is critical for the biosynthesis of the mugineic acid family of phytosiderophores in graminaceous plants, and for the homeostasis of metal ions in nongraminaceous plants. We isolated one genomic NAS clone, ZmNAS3, and two cDNA NAS clones, ZmNAS1 and ZmNAS2, from maize (Zea mays cv Alice). In agreement with the increased secretion of phytosiderophores with Fe deficiency, ZmNAS1 and ZmNAS2 were positively expressed only in Fe-deficient roots. In contrast, ZmNAS3 was expressed under Fe-sufficient conditions, and was negatively regulated by Fe deficiency. This is the first report describing down-regulation of NAS gene expression in response to Fe deficiency in plants, shedding light on the role of nicotianamine in graminaceous plants, other than as a precursor in phytosiderophore production. ZmNAS1-green fluorescent protein (sGFP) and ZmNAS2-sGFP were localized at spots in the cytoplasm of onion (Allium cepa) epidermal cells, whereas ZmNAS3-sGFP was distributed throughout the cytoplasm of these cells. ZmNAS1 and ZmNAS3 showed NAS activity in vitro, whereas ZmNAS2 showed none. Due to its duplicated structure, ZmNAS2 was much larger (65.8 kD) than ZmNAS1, ZmNAS3, and previously characterized NAS proteins (30-38 kD) from other plant species. We reveal that maize has two types of NAS proteins based on their expression pattern and subcellular localization.     

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.     

Promoter analysis of iron-deficiency-inducible barley IDS3 gene in Arabidopsis and tobacco plants.

Under conditions of iron deficiency, graminaceous plants induce the expression of genes involved in the biosynthesis of mugineic acid family phytosiderophores. We previously identified the novel cis-acting elements IDE1 and IDE2 (iron-deficiency-responsive element 1 and 2) through promoter analysis of the barley (Hordeum vulgare L.) iron-deficiency-inducible IDS2 gene in tobacco (Nicotiana tabacum L.). To gain further insight into plant gene regulation under iron deficiency, we analyzed the barley iron-deficiency-inducible IDS3 gene, which encodes mugineic acid synthase. IDS3 promoter fragments were fused to the beta-glucuronidase (GUS) gene, and this construct was introduced into Arabidopsis thaliana L. and tobacco plants. In both Arabidopsis and tobacco, GUS activity driven by the IDS3 promoter showed strongly iron-deficiency-inducible and root-specific expression. Expression occurred mainly in the epidermis of Arabidopsis roots, whereas expression was dominant in the pericycle, endodermis, and cortex of tobacco roots, resembling the expression pattern conferred by IDE1 and IDE2. Deletion analysis revealed that a sequence within -305 nucleotides from the translation start site was sufficient for specific expression in both Arabidopsis and tobacco roots. Gain-of-function analysis revealed functional regions at -305/-169 and -168/-93, whose coexistence was required for the induction activity in Arabidopsis roots. Multiple IDE-like sequences were distributed in the IDS3 promoter and were especially abundant within the functional region at -305/-169. A sequence moderately homologous to that of IDE1 was also present within the -168/-93 region. These IDE-like sequences would be the first candidates for the functional iron-deficiency-responsive elements in the IDS3 promoter.