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.     

Characterization of phytosiderophore secretion under Fe deficiency stress in Festuca rubra

In the present study we investigated the response to iron (Fe) deficiency in two cultivars of Festuca rubra L. (Rubina and Barnica) used in correction of chlorosis of fruit trees cultivated on calcareous soils. We found that a Fe-chelating compound, identified as 2-deoxymugineic acid (DMA), was secreted from the roots in response to Fe-deficiency in both cultivars. The amount of DMA secreted into solution increased with the development of Fe-deficiency. The secretion showed a distinct diurnal rhythm characterized by a secretion peak at between 2 and 5 hours after sunrise at 20°C. However, this secretion peak was delayed by 3 hour at low temperature (<10°C) and occurred 3 h earlier at high temperature (30°C). When water used for the collection of root exudates was pre-warmed (25°C) or pre-cooled (10°C), this led to an earlier or a delayed secretion compared to control (15°C) under the same air temperature, respectively. Short-term shading treatment did not affect the secretion pattern of DMA. These results demonstrate that the secretion time of DMA from the roots is, at least partly controlled by the temperature in the root environment. Overall, these findings suggest that the ability of Festuca rubra to prevent Fe chlorosis symptoms (`re-greening effect') of associated fruit trees is partially related to the secretion of DMA which increase Fe availability in calcareous soils.     

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     

Phytosiderophore release in relation to micronutrient metal deficiencies in barley

Phytosiderophore release occurs under both iron and zinc deficiencies in representative Poaceae and has been speculated to be a general adaptive response to enhance the acquisition of micronutrient metals. To test this hypothesis, phytosiderophore (PS) release rates from barley (Hordeum vulgare cv. CM72) subjected to deficiencies of Fe, Zn, Mn, and Cu were compared using chelator-buffered nutrient solutions. PS release rates were determined at two day intervals during onset and development of deficiency symptoms. Plant dry matter yields and nutrient concentrations, measured at three time points were used to construct growth curves for calculation of PS release per unit root mass and estimation of critical internal nutrient levels associated with PS release. In comparison to trace metal-sufficient control plants, dry matter production was markedly reduced in the Zn, Mn, and Cu deficiency treatments, with final relative yields of 49, 61, and 34%, respectively. Relative yields for Fe-deficient plants grown at three suboptimal Fe levels ranged from 95 to 33% of control, and provided a basis for comparison of PS release rates by Zn-, Mn-, and Cu-deficient plants at similar levels of growth inhibition. Under Fe deficiency, PS release increased with severity of the deficiency as measured by foliar Fe concentration, yield reduction, and chlorosis. Changes in PS release rates over time suggested a cyclical pattern that may be regulated by Fe concentration in the plant shoot. The highest rate of PS release (35 mol g^–1 root dw 2 h^–1) was measured after 10 days of growth at pFe 19, whereas control plants adapted for growth at pFe 17 released only 2 to 3 mol g^–1 root dw 2 h^–1. In a second experiment, maximum PS release rates for barley subjected to Zn, Mn, and Cu deficiencies were only 2.6, 2.5 and 1 mol g^–1 2 h^–1, respectively and were only slightly elevated over those of the control plants (ca. 1 mol g^–1 root dw 2 h^–1) grown at pFe 16.5. Moreover, enhanced PS release under Zn deficiency occurred much later, after the deficiency had already caused severely reduced growth. The results suggest that phytosiderophore release in this barley cultivar is a specific response to Fe deficiency and is not significantly induced in response to deficiencies of other trace metals.     

Comparative evaluation of iron solubilizing substances (phytosiderophores) released by oats and corn: Iron-efficient and iron-inefficient plants

Release of phytosiderophores from barley (Hordeum vulgare L.) in response to Fe-deficiency stress prompted further testing of other graminaceous (grass) species for phytosiderophore release and results have prompted characterization of these plants into a Strategy II designation. This classification denotes an enhanced release of phytosiderophore in response to Fe-deficiency stress with a concomitant uptake of Fe by the plant. The objective of this study was to determine if Fe-inefficient and Fe-efficient corn (Zea mays L.) differ in their release of Fe solubilizing substances in response to Fe-deficiency stress. We have not identified the specific structure of these substances but refer to them as phytosiderophores to further characterize their behavior. By our indirect method, there was no measurable release of Fe solubilizing substances (phytosiderophores) from either the Fe-efficient WF9 or the Fe-inefficient ys₁ corn despite WF9 being greener and apparently more Fe efficient than ys₁. Fe-efficient Coker 227 oats (Avena byzantina C. Koch.) has been found to release a phytosiderophore whereas the Fe-inefficient TAM 0-312 does not. Iron-stressed Coker 227 oats released Fe solubilizing substances when grown in the same solution with WF9 corn which resulted in greening and Fe uptake by WF9 corn. Iron efficiency in these two graminaceous species appears to be controlled by different mechanisms.     

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.     

Identification of two novel phytosiderophores secreted by perennial grasses

It has been suggested that some perennial grasses secrete phytosiderophores in response to iron (Fe) deficiency, but the compounds have not been identified. Here, we identified and characterized the phytosiderophores secreted by two perennial grasses, Lolium perenne cv. Tove and Poa pratensis cv. Baron. Root exudates were collected from the roots of Fe-deficient grasses and then purified with various chromatographies. The structure of the purified compounds was determined using both nuclear magnetic resonance and fast atom bombardment mass spectrometry. Both species secreted phytosiderophores in response to Fe deficiency, and the amount of phytosiderophores secreted increased with the development of Fe deficiency. The type of phytosiderophores secreted differed with plant species; L. perenne cv. Tove secreted 3-epihydroxy-2'-deoxymugineic acid (epiHDMA), 2'-deoxymugineic acid (DMA) and an unknown compound, whereas P. pratensis cv. Baron secreted DMA, avenic acid A (AVA) and an unknown compound. Purification and subsequent analysis with nuclear magnetic resonance and mass led to identification of the two novel phytosiderophores; 3-hydroxy-2'-deoxymugineic acid (HDMA) from L. perenne, and 2'-hydroxyavenic acid A (HAVA) from P. pratensis. Both novel phytosiderophores have similar chelating activity to known phytosiderophores.     

Light-mediated release of phytosiderophores in wheat and barley under iron or zinc deficiency

The effect of varied light intensity (50 – 600 mol m-2 s-1) on the rate of phytosiderophore release was studied under zinc (Zn) deficiency using a bread (Triticum aestivum L. cv. Aroona) and a durum wheat cultivar (Triticum durum Desf. cv. Durati) differing in zinc (Zn) efficiency and under iron (Fe) deficiency using a barley cultivar (Hordeum vulgare L. Europe). Plants were grown under controlled environmental conditions in nutrient solution for 15 days (wheat plants) or 11 days (barley plants). Phytosiderophore release was determined by measuring capacity of root exudates to mobilize copper (Cu) from a Cu-loaded resin.With increasing light intensity visual Zn deficiency symptoms such as whitish-brown lesions on leaf blade developed rapidly and severely in wheat, particularly in the durum cultivar Durati. In wheat plants supplied well with Zn, increases in light intensity from 100 to 600 mol m-2 s-1 did not clearly affect the rate of phytosiderophore release. However, under Zn deficiency increases in light intensity markedly enhanced release of phytosiderophores in Zn-deficient Aroona, but not in Zn-inefficient Durati. When Fe-deficient barley cultivar Europe was grown first at 220 mol m-2 s-1 and then exposed to 600 mol m-2 s-1 for 24 and 48 h, the rate of release of phytosiderophores was enhanced about 4-fold and 7-fold, respectively. Transfer of Fe-deficient plants from 600 to 50 mol m-2 s-1 for 48 h reduced the rate of release of phytosiderophores by a factor of 7. The effect of light on phytosiderophore release was similar regardless of whether the rate of phytosiderophore release was expressed per plant or per unit dry weight of roots.The results demonstrate a particular role of light intensity in phytosiderophore release from roots under both Zn and Fe deficiency. It is suggested that in the studies concerning the role of phytosiderophore release in expression of Zn or Fe efficiency among and within cereals, a special attention should be given to the light conditions.     

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.