Senescence and protein remobilisation in leaves of maturing wheat plants grown on waterlogged soil

Nicotianamine (NA), the key precursor of the mugineic acid family phytosiderophores (MAs), is synthesized from S-adenosylmethionine (SAM). The NA synthase was strongly induced by Fe-deficiency treatment, and the activity increased to the maximum level faster than the time of maximum level of MAs secretion and also before the appearance of severest chlorosis. The enzyme was mainly localized in the roots of barley. NA synthase had the optimum pH at 9.0, a molecular weight of about 40,00050,000 estimated by gel filtration or about 30,000 by SDS-PAGE. Using hydrophobic chromatography, hydroxylapatite chromatography, and preparative SDS-PAGE, NA synthase was purified as one band on SDS-PAGE.     

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     

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     

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 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.     

Biosynthesis of Phytosiderophores : In Vitro Biosynthesis of 2'-Deoxymugineic Acid from l-Methionine and Nicotianamine

2′ -Deoxymugineic acid (DMA), one of mugineic acid-family phytosiderophores (MAs), was synthesized in vitro both from l-methionine and from nicotianamine (NA) with a cell-free system derived from root tips of iron-deficient barley (Hordeum vulgare L.). The reactions producing DMA from NA needed an amino group acceptor (i.e. 2-oxoglutarate, pyruvate, or oxalacetic acid) and a reductant (i.e. NADH or NADPH). The activity of the enzymes to produce NA from l-methionine was the highest at about pH 9. This biosynthetic activity was markedly induced by iron-deficiency stress. The synthesis of NA from S-adenosyl-l-methionine was more efficient than from l-methionine. From the results with the cell-free system reported here, we propose a revised biosynthetic pathway of MAs.     

Development and Characterization of a Monoclonal Antibody to Phytosiderophores

Mugineic acid-family phytosiderophores (MAs) are low molecularweight chelators that are secreted by graminaceous plants, formcomplexes with soil Fe(III) and are essential for plant growth.Methods to detect MAs which include HPLC and radio-immunoassaywith polyclonal antibody require sophisticated equipment orradio-labelled MAs which are difficult to synthesize. Our objectivewas to develop a detection and quantitation system for MAs basedon monoclonal antibody specificity and technology. A monoclonalantibody was produced which reacts with nicotianamine (NA),deoxymugineic acid (DMA), mugineic acid (MA) and epi-hydroxymugineicacid (epi-HMA) in a competitive ELISA. Azetidine-2-carboxylicacid (A-2-C) was not reactive while N-(3-amino-3-carboxypropyl)azetidine-2-carboxylic acid (A-2-C dimer) was partially reactive.The range of detection using the competitive ELISA is from 2x 10^–6 to 2 x 10^–7 M MAs. Besides detection andquantification of MAs, the potential uses for the monoclonalantibody are numerous and include affinity chromatography andimmunocytochemistry. (Received September 26, 1991; Accepted December 16, 1991)     

Identification and molecular characterization of the nicotianamine synthase gene family in bread wheat

Nicotianamine (NA) is a non‐protein amino acid involved in fundamental aspects of metal uptake, transport and homeostasis in all plants and constitutes the biosynthetic precursor of mugineic acid family phytosiderophores (MAs) in graminaceous plant species. Nicotianamine synthase (NAS) genes, which encode enzymes that synthesize NA from S‐adenosyl‐L‐methionine (SAM), are differentially regulated by iron (Fe) status in most plant species and plant genomes have been found to contain anywhere from 1 to 9 NAS genes. This study describes the identification of 21 NAS genes in the hexaploid bread wheat (Triticum aestivum L.) genome and their phylogenetic classification into two distinct clades. The TaNAS genes are highly expressed during germination, seedling growth and reproductive development. Fourteen of the clade I NAS genes were up‐regulated in root tissues under conditions of Fe deficiency. Protein sequence analyses revealed the presence of endocytosis motifs in all of the wheat NAS proteins as well as chloroplast, mitochondrial and secretory transit peptide signals in four proteins. These results greatly expand our knowledge of NAS gene families in graminaceous plant species as well as the genetics underlying Fe nutrition in bread wheat.     

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.     

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.     

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.     

Nicotianamine synthase 2 localizes to the vesicles of iron‐deficient rice roots,and its mutation in the YXXφ or LL motif causes the disruption of vesicle formation or movement in rice

Graminaceous plants release mugineic acid family phytosiderophores (MAs) to acquire iron from the soil. Here, we show that deoxymugineic acid (DMA) secretion from rice roots fluctuates throughout the day, and that vesicles accumulate in roots before MAs secretion. We developed transgenic rice plants that express rice nicotianamine (NA) synthase (NAS) 2 (OsNAS2) fused to synthetic green fluorescent protein (sGFP) under the control of its own promoter. In root cells, OsNAS2–sGFP fluorescence was observed in a dot‐like pattern, moving dynamically within the cell. This suggests that these vesicles are involved in NA and DMA biosynthesis. A tyrosine motif and a di‐leucine motif, which have been reported to be involved in cellular transport, are conserved in all identified NAS proteins in plants. OsNAS2 mutated in the tyrosine motif showed NAS activity and was localized to the vesicles; however, these vesicles stuck together and did not move. On the other hand, OsNAS2 mutated in the di‐leucine motif lost NAS activity and did not localize to these vesicles. The amounts of NA and DMA produced and the amount of DMA secreted by OsNAS2–sGFP plants were significantly higher than in non‐transformants and domain‐mutated lines, suggesting that OsNAS2–sGFP, but not the mutated forms, was functional in vivo. Overall, the localization of NAS to vesicles and the transport of these vesicles are crucial steps in NA synthesis, leading to DMA synthesis and secretion in rice.     

Iron acquisition by plants.

In nongraminaceous plants, the FeII-transporter gene and ferric-chelate reductase gene have been cloned from Arabidopsis thaliana, whereas FeIII-reductase has not. In graminaceous monocots, the genes for mugineic acids (MAs) synthesis, nas (nicotianamine synthase) and naat (nicotianamine aminotransferase), have been cloned from barley, whereas the FeIII-MAs transporter gene is yet to be cloned. Transferrin absorption in Dunaliella has been reported, suggesting a phagocytotic (endocytotic) Fe-acquisition mechanism. Work to develop transgenic cultivars tolerant to Fe-deficiency in calcareous soils is now in progress.     

Biosynthesis of nicotianamine in the suspension-cultured cells of tobacco (Nicotiana megalosiphon)

Summary Seven kinds of suspension cell cultures from five species ofNicotiana were screened for the occurrence of nicotianamine. Nicotianamine was detected in the cultured cells ofN. megalosiphon andN. plumbaginifolia.l-[l-¹⁴C]Methionine, which is the precursor of the mugineic-acid-family phytosiderophores and nicotianamine in barley plants, was incorporated into nicotianamine by the cultured cells ofN. megalosiphon both in vivo and in vitro. The advantage of the cultured tobacco cells for the study of the biosynthesis of nicotianamine and the mugineic-acid-family phytosiderophores is discussed.     

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     

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.     

Biosynthesis of avenic acid A in oat cv. Onward: studies with 14C or 15N labeled compounds

The precursory role of avenic acid A (AVA) in the biosynthesis of the mugineic acid family (MAs) of phytosiderophores was studied by feeding ¹⁴C or ¹⁵N labeled compounds into iron-deficient oat roots (Avena sativa L. cv. Onward). Carbon-14 of methionine was incorporated into AVA and 2-deoxymugineic acid (DMA) in the oat roots, while ¹⁴C of homoserine was not incorporated into either AVA or DMA. The molar radioactivity of DMA was higher than that of AVA. Incorporation of ¹⁵N into MAs was examined by feeding ¹⁵N-ammonium sulfate into oat roots. The value of ¹⁵N atom-% excess of DMA was higher than that of AVA.These results indicate that methionine, rather than homoserine, is the direct precursor of MAs in oat, which is similar to that in barley, and that AVA is not the precursor of the other MAs.