Fe-deficient Barley Roots Research Articles

Time course analysis of gene expression over 24 hours in Fe-deficient barley roots

Typical for a graminaceous plant, barley secretes mugineic acid-family phytosiderophores (MAs) to acquire iron (Fe). Under Fe-deficient conditions, MAs secretion from barley roots increases markedly. Secretion shows a diurnal pattern, with a clear peak 2-3 h after sunrise and cessation within a few hours. Microarray analyses were performed to profile the Fe deficiency-inducible genes in barley roots and diurnal changes in the expression of these genes. Genes encoding enzymes involved in MAs biosynthesis, the methionine cycle, and methionine biosynthesis were highly induced by Fe deficiency. The expression of sulfate transporters was also upregulated by Fe deficiency. Therefore, all of the genes participating in the MAs pathway from sulfur uptake and assimilation to the biosynthesis of MAs were upregulated in Fe-deficient barley roots. In contrast to MAs secretion, the transcript levels of these genes did not show diurnal changes. The amount of endogenous MAs gradually increased during the day after MAs secretion ceased, and was highest before secretion began. These results show that MAs biosynthesis, including the supply of the substrate methionine, occurs throughout the day, and biosynthesized MAs likely accumulate in barley roots until their secretion into the rhizosphere. In contrast, the levels of transcripts encoding an Fe(III)-MAs complex transporter, two putative metal-MAs complex transporters, and HvYS1 were also increased in Fe-deficient barley roots, and the levels of two of these transcripts showed diurnal rhythms. The Fe(III)-MAs complex transporters may absorb Fe(III)-MAs diurnally, synchronous with the diurnal secretion of MAs.

Biosynthesis and secretion of mugineic acid family phytosiderophores in zinc‐deficient barley

Mugineic acid family phytosiderophores (MAs) are metal chelators that are produced in graminaceous plants in response to iron (Fe) deficiency, but current evidence regarding secretion of MAs during zinc (Zn) deficiency is contradictory. Our studies using HPLC analysis showed that Zn deficiency induces the synthesis and secretion of MAs in barley plants. The levels of the HvNAS1, HvNAAT-A, HvNAAT-B, HvIDS2 and HvIDS3 transcripts, which encode the enzymes involved in the synthesis of MAs, were increased in Zn-deficient roots. Studies of the genes involved in the methionine cycle using microarray analysis showed that the transcripts of these genes were increased in both Zn-deficient and Fe-deficient barley roots, probably allowing the plant to meet its demand for methionine, a precursor in the synthesis of MAs. In addition, HvNAAT-B transcripts were detected in Zn-deficient shoots, but not in those that were deficient in Fe. Increased synthesis of MAs in Zn-deficient barley was not due to a deficiency of Fe, because Zn-deficient barley accumulated more Fe than did the control plants, ferritin transcripts were increased in Zn-deficient plants, and Zn deficiency promoted Fe transport from root to shoot. Moreover, analysis using the positron-emitting tracer imaging system (PETIS) confirmed that more 62Zn(II)-MAs than 62Zn2+ were absorbed by the roots of Zn-deficient barley plants. These data suggest that the increased biosynthesis and secretion of MAs arising from a shortage of Zn are not due to an induced Fe deficiency, and that secreted MAs are effective in absorbing Zn from the soil.

Isolation and characterization of IDI2, a new Fe-deficiency-induced cDNA from barley roots, which encodes a protein related to the alpha subunit of eukaryotic initiation factor 2B (eIF2Balpha).

A new Fe-deficiency-inducible cDNA, IDI2, was isolated from Fe-deficient barley roots using the cDNA MACRO Array Technique. Accumulation of IDI2 transcripts in barley roots was strongly correlated with iron nutritional status. IDI2 encoded a protein with a low similarity to the alpha subunit of eukaryotic initiation factor 2B (eIF2Balpha). In addition, many hypothetical proteins homologous to IDI2 were also found in a database search. These proteins had limited similarity to eIF2Balpha as well as IDI2. It has been reported that these eIF2Balpha-like proteins (eIF2Balpha-LPs) are a family that is distinct from the eIF2Balpha/beta/delta family and widely distributed in the archaea, bacteria, and eukarya. A phylogenic analysis revealed that IDI2 is the first member of the eIF2Balpha-LP family to be found in higher plants. A possible role of IDI2 protein in regulating protein synthesis in Fe-deficient barley roots is proposed.

Induction of the IDI1 gene in Fe-deficient barley roots: A gene encoding a putative enzyme that catalyses the methionine salvage pathway for phytosiderophore production

IDI1, a cDNA induced by Fe-deficiency, was isolated from Fe-deficient barley roots by differential hybridization screening. IDI1 transcripts in roots increased with Fe-deficiency treatment. IDI1 cDNA encoded a protein with 198 amino acids. The IDI1 protein is homologous to the bacterial protein E-2, which catalyzes the formation of a 2-keto-methylthiobutyric acid in the methionine salvage pathway. IDI1 protein is postulated to be an enzyme that participates in the pathway providing methionine for phytosiderophore production in Fedeficient barley roots.

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.

Presence of nicotianamine synthase isozymes and their homologues in the root of graminaceous plants

Nicotianamine synthase (NAS) catalyzes the synthesis of nicotianamine, which is an intermediate in the biosynthetic pathway of mugineic acid family phytosiderophores (MAs). Using polyclonal anti-NAS antibodies and recombinant NAS proteins, we identified five NAS isozymes and one NAS homologue in Fe-deficient barley roots using two-dimensional electrophoresis followed by Western blot analysis. Other unidentified NAS homologues that were induced by Fe-deficiency were also detected in barley roots. Western analysis enabled to detect NAS homologues in wheat, oats, rice, maize, and sorghum roots. In graminaceous species, both the amount and number of NAS homologues were correlated with the total NAS activity and Fe-deficiency tolerance. The NAS isoform patterns differed among the graminaceous plants.

Cloning of nicotianamine synthase genes, novel genes involved in the biosynthesis of phytosiderophores.

Nicotianamine synthase (NAS), the key enzyme in the biosynthetic pathway for the mugineic acid family of phytosiderophores, catalyzes the trimerization of S-adenosylmethionine to form one molecule of nicotianamine. We purified NAS protein and isolated the genes nas1, nas2, nas3, nas4, nas5-1, nas5-2, and nas6, which encode NAS and NAS-like proteins from Fe-deficient barley (Hordeum vulgare L. cv Ehimehadaka no. 1) roots. Escherichia coli expressing nas1 showed NAS activity, confirming that this gene encodes a functional NAS. Expression of nas genes as determined by northern-blot analysis was induced by Fe deficiency and was root specific. The NAS genes form a multigene family in the barley and rice genomes.

Formate dehydrogenase, an enzyme of anaerobic metabolism, is induced by iron deficiency in barley roots.

To identify the proteins induced by Fe deficiency, we have compared the proteins of Fe-sufficient and Fe-deficient barley (Hordeum vulgare L.) roots by two-dimensional polyacrylamide gel electrophoresis. Peptide sequence analysis of induced proteins revealed that formate dehydrogenase (FDH), adenine phosphoribosyltransferase, and the lds3 gene product (for Fe deficiency-specific) increased in Fe-deficient roots. FDH enzyme activity was detected in Fe-deficient roots but not in Fe-sufficient roots. A cDNA encoding FDH (Fdh) was cloned and sequenced. Fdh expression was induced by Fe deficiency. Fdh was also expressed under anaerobic stress and its expression was more rapid than that induced by Fe deficiency. Thus, the expression of Fdh observed in Fe-deficient barley roots appeared to be a secondary effect caused by oxygen deficiency in Fe-deficient plants.

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

Two isozymes of nicotianamine aminotransferase, involved in the biosynthesis of phytosiderophores, were detected in Fe-deficient barley roots. They showed different molecular masses and kinetic parameters. Furthermore, one of them was strongly induced by an Fe-deficiency treatment, whereas the other was not.

Purification and characterization of nicotianamine synthase from Fe-deficient barley roots

Purification and characterization of nicotianamine synthase from Fe-deficient barley roots

Expression of a Gene Specific for Iron Deficiency (<italic>Ids3</italic>) in the Roots of <italic>Hordeum vulgare</italic>

To clone genes required for the synthesis of mugineic acid (MA) or for the transport of Fe(III)-MA, a lambda ZAPII cDNA library was constructed from poly(A)(+)-RNA isolated from Fe-deficient barley roots. The cDNA library was then used for differential screening of barley roots that had been grown in the presence and absence of iron. Seven clones that hybridized specifically to the probe for Fe deficiency were selected. One clone, presumably encoding a full-length mRNA, as deduced from Northern hybridization, was sequenced. The clone consisting of 1685 nucleotides encoded a putative protein of 169 amino acids and an M(r) of 18704. The gene was specifically expressed in the roots of iron-deficient barley. A search for homologies in a protein database (NBRF) revealed that the predicted protein product has a functional peptide domain that resembles that of 2-oxoglutarate-dependent dioxygenases.

An iron deficiency-specific cDNA from barley roots having two homologous cysteine-rich MT domains

For iron acquisition grasses secrete iron chelators called mugineic acid-family phytosiderophores (MAs) which solubilize sparingly soluble Fe 3 +, viz. Fe(OH)3, in the rhizosphere, and reabsorb Fe(III)-MAs complex [1] thus formed. Under Fe-deficient conditions the secreted amount of MAs and the absorption rate of the Fe(III)-MAs complex accelerates [2]. At the same time some peptides in the roots also increase [3]. So, we tried to isolate and characterize cDNA clones involved in iron acquisition, i.e. the genes of MAs biosynthesis and also the Fe(III)-MAs transporter. These proteins or enzymes have neither been identified nor isolated to date. So, inevitably, we could not help selecting several cDNA clones specific to Fe-deficient barley roots by differential hybridization. First, a 2gtl0 cDNA library was constructed with mRNA isolated from Fe-deficient barley roots. Then the library was differentially screened between cDNA probes from mRNA of Fedeficient and Fe-sufficient barley roots. Altogether, seven cDNA clones which hybridized specifically to a probe of Fe-deficient barley roots were selected. Their inserts were subcloned at the Eco RI site of pBluescriptlI SK + vector, and then DNAs were sequenced. But their inserts were short (100-500 bp) and were not likely to include the full length of mRNA. Therefore we next screened a newly constructed 2zaplI cDNA library of Fe-deficient barley roots using one of the seven clones as a probe. Twenty-four clones were subcloned at the Eco RI and Xho I sites of the pBluescriptlI SKvector. The longest clone of about 500 bp was assumed to have the full length by northern hybridization. We named this clone ids-1 (iron deficiency-specific clone 1) (Fig. 1). The sequenced ids-1 is composed of 503 nucleotides, a putative open reading frame of 222 bp, a 5'-untranslated sequence of 31 bp and a 3' -noncoding region of 250 bp. A 3' -noncoding region contains the sequence AATATA 23 bp upstream from the polyadenylation site which is homologous to polyadenylation signals (AATAAA). There are many sequences of palindrome, hairpin loop and tandem repeats. The putative open reading frame encodes a protein of 74 residues (7500 Da) which contains two cysteine-rich regions. Comparison of this predicted protein with the NBRF protein database (DNASIS software) shows exclusively selected MT sequences. The strong similarity of the ids-1 sequence to animal and fungal MTs is in two domains. Each domain contains six cysteines arranged as Cys-X-Cys clusters (Fig. 1), a presumable constraint of metal-binding characteristic of MTs [4]. In the plant world similar domains are also

Characterization of mugineic-acid-Fe transporter in Fe-deficient barley roots using the multi-compartment transport box method

We have investigated the mugineicacid-Fe transport activity of Fe-deficient barley roots, using the multi-compartment transport box system. The roots maintained Fe transport activity for 20 h after excision. The following results were obtained. (1) In Fe-deficient roots, mugineic acid addition enhanced the transport of Fe by 32.2 times over that of the control (with FeC13 addition). (2) The mugineic-acid-55Fe transport activity of Fe-deficient roots was 18.4-fold higher than that of the Fe-sufficient roots. (3) The mugineic-acid-55Fe transport activity was decreased (7.13% based on the control) by treatment with 5 μM carbonylcyanidem-chlorophenyl hydrazone (CCCP). Pretreatment with 0.1 mM dicyclohexyl carbodiimide (DCCD) lowered the transport activity (10.7% based on the control) and 1 mMN-ethylmaleimide (NEM) pretreatment reduced the transport activity to a value equivalent to 2.41% of that in the control. It is concluded that mugineicacid-Fe transporter is induced in its activity and/or amount by Fe-deficiency treatment and has an SH residue at its active site, and that the transporter needs the proton motive force produced by ATPase. We detected three polypeptides (14, 28 and 40 kDa) in the root plasma membrane that were induced under Fe-deficiency treatment.