The Isolation and Characterization of the IronPhytosiderophore Transporter in Avena sativa A thesis submitted to the School of Graduate Studies of Bloomsburg University In Partial fulfillment of the Requirement For the Degree of Master of Science Program in Biology Department of Biological and Allied Health Sciences Bloomsburg University of Pennsylvania By Melissa L. Tomcavage Bloomsburg,Pennsylvania 2009 Masters Candidate This Thesis was accepted as meeting the research requirement for the master's degree. APPROVED: , J.--........_ Date: :)7 LY-/22 ..9 .p/.m 1ilb&J- Date: )~h/~ Date: ) / _5' Dr. Kristen D. Brubaker, Committee Member Lft_Ej Dr. Kevin J. Williams, Committee Member APPROVED: Date: 6~ ~ Dr. L ~ & G r a d u a t e Studies 11 Abstract Iron is a vital nutrient required for processes such as photosynthesis and respiration in plants. Unfortunately, the mineral is found as an insoluble form in the rhizosphere. Plants have evolved two different mechanisms for acquiring iron, a reduction based strategy and a chelation based strategy. The chelation based strategy is restricted to grass species as they are the only plants found to put out phytosiderophores. Until recently, the mechanisms for the uptake of the phytosiderophore complexed to its ligand has been unknown. The discovery of the membrane transporter gene used by the phytosiderophore in maize in 200 I has shed some light on the mechanisms. A second transporter gene was discovered in 2006 in barley that moves ironphytosiderophore complexes into the root. This thesis presents the data for the sequence of a putative iron-phytosiderophore transporter gene in oats that was compared to the sequences of known phytosiderophore transporters, HvYS 1 from barley and ZmYS 1 from maize. ll1 Table of Contents Abstract lll Table of contents lV List of Figures V List of Tables Vl Acknowledgements vu Introduction 1 Iron Nutrition 1 Iron Nutrition in Plants 2 Iron Uptake Mechanisms of Plants 5 Phytosiderophores 7 Mugineic Acid 8 Phytosiderophore Secretion 11 Yellow Stripe 1 13 Experimental Approach 17 Results 21 Discussion 34 References 37 lV List of Figures Figure 1. Strategy I Uptake. 6 Figure 2. Strategy II Uptake 7 Figure 3. Oats grown hydroponically under iron deficient conditions. 17 Figure 4. Oat roots in iron deficient hydroponic solution. 17 Figure 5. Specificity Region of Agrostis stolonifera. 22 Figure 6. Nucleotide BLAST of A. stolonifera specificity region. 22-23 Figure 7. Nucleotide BLAST of A. stolonifera specificity region. 23 Figure 8: Amino Acid sequence of A. stolonifera specificity region. 24 Figure 9: Clustal Alignment of specificity regions and known transporters. 24 Figure 10. Full gene analysis, Nucleotide sequence. 27 Figure 11: Full gene nucleotide BLAST of Avena putative transporter. 28-30 Figure 12: Full gene protein analysis of putative Avena transporter. 30 Figure 13: Full Gene Protein BLAST of putative Avena transporter. 31 Figure 14. Clustal analysis between known transporter and putative Avena tranporter. 32-33 V List of Tables Table 1. Primers 20 VI Acknowledgements I would like to thank my thesis advisor, Dr. George Davis, for his guidance during the course of this project. Input from Dr. Kristen Brubaker was highly appreciated as well. Thanks to Dr. John Hranitz for his help with initial sequencing and to Dr. Carl Hansen for help with bioinformatics analysis. In addition to the faculty, I thank several peers for assistance during various parts of this project: Joshua Montgomery and Shirshendu Saha, assistance with the benchwork; Andrew Troutman, assistance with initial sequencing; Essie Reed and Heather Pursel, assistance in obtaining plant material. I would also like to acknowledge Gene Wiz, Inc. for performing sequencing runs. This project was funded by grants from the Department of Agriculture and the Internal Grant, ####, from Bloomsburg University. The US Department of Agriculture, CS REES grant is as follows: Project number: 2007-35318-18350 Project Title: Uptake Specificity of Snythetic Phytosiderophore Analogs by Graminaceous Plants. Vll Introduction Iron Nutrition According to the World Health Organization, iron deficiency is the most common and widespread nutritional disorder in the world (www.who.int). Humans depend mostly on plant foods for nutritional health and plants are critical components of our diet in that they provide almost all essential mineral and organic nutrients either directly or indirectly. A deficiency in the agricultural realm leads to reduced crop yields as well (Astolfi et al., 2003; Grusak and DellaPenna 1999). Mammalian systems contain at least four major classes of iron-containing proteins: iron containing enzymes which include hemoglobin, myoglobin, and cytochromes; iron-sulfur enzymes such as flavoproteins and heme-flavoproteins; proteins for iron storage and transport (ferritins); and other iron-containing or activated enzymes (Vasconcelos and Grusak 2006). Nutritional iron is divided into heme iron (easily absorbed) and non-heme iron (usually considered "free" or "weak"). Heme iron intake is negligible for the majority of people in many developing countries based on a more vegetarian type of diet. Therefore, non-heme iron is the main source of dietary iron for most people in the world. Iron in any food has a particular bioavailability, which is a function of its chemical form and the presence of other food components that either promote or inhibit its absorption. The iron present in the human body is mostly in a stored form and losses are usually minimal. However, dietary intake of iron is needed to replace the iron lost by passage of stool and urine, shedding of skin, and sweating (Vasconcelos and Grusak 2006). Plants contain almost all essential human nutrients, with cereal grains such as wheat, rice, and maize providing about two-thirds of all energy in the human diet. However, not all plant sources provide the same amount of iron, and the amount of iron ingested is directly proportional to the portion size that is consumed (Vasconcelos and Grusak 2006; Cassman 1999). Iron acquisition is challenging due to the low availability ofiron in soil. Although iron is the fourth most abundant element in the earth's crust, it is not readily available to plants because it is found in most rocks as primary minerals in ferromagnesium silicate forms like olivine, augite, hornblende, or biotite, which are considered unstable. One third of the 1 cultivated soils around the world are calcareous and therefore iron deficient (Kim and Guerinot 2007; Brown and Jolley 1989; Briat 2008; Ma 2005). The total Fe2O3 content of soils varies from about 2-6% in nonnal temperate soil to as much as 60% in tropical soils (Steward 1963). Other factors such as high calcium and magnesium concentrations and high bicarbonate concentrations, soil carbonates, salinity, soil iron composition nutrient interaction, soil moisture content, and soil bulk density also impact iron bioavailability (Ma 2005; Murata et al., 2006; Hansen et al., 2006). The Industrial Revolution caused an increase in food production which led to agricultural practices involving application of chemical fertilizers, manures, pesticides, and other synthetic products to maximize output. These practices caused an alteration in soil characteristics, such as soil pH, nutritional conditions, and soil water conditions. The result is serious mineral imbalances and heavy metal contamination in agricultural fields all over the world (Hiradate et al. 2007). Iron Nutrition in Plants If a continuous supply of iron from the soil is not available, the plant develops iron chlorosis - a yellowing caused by low chlorophyll content in leaves that can be alleviated by supplying the plant with iron (Astolfi et al. 2003; Brown and Jolley 1989; Briat 2008; Ma 2005; Thoiron et al. 1997). Chlorosis occurs in the young leaves, with the other leaves frequently unaltered. Iron cannot be withdrawn from mature leaves to support growth of new leaves and the veins of the leaves maintain their chlorophyll while the interveinous regions lose theirs, giving the plant a characteristic appearance of iron deficiency. The effects of iron deficiency are often associated with cytological alterations which mainly affect the chloroplast ultrastructure. Thylakoids become disorganized and most of the mitochondria located in chlorotic leaves display completely disorganized cristae (Briat 2008; Epstein 1972; Scott 2008; Thoiron et al. 1997). Iron is required in chlorophyll biosynthesis; iron deficiency impacts chlorophyll degradation by increasing it - the quantity of carotenoids decreases when iron is in short supply, resulting in chlorophy 11 degradation (Briat 2008). Once total bleaching occurs in severe conditions of iron deficiency, leaf margins collapse or whole leaves in general take on a "papery" attribute (Steward 1963). Problems 2 related to the low solubility of iron bearing mineral phases, slow dissolution kinetics, the transport of soluble iron species to the root, the low bioavailability of certain soluble iron complexes, and slow iron release from complexes in ligand or metal exchange reaction need to be overcome in order to supply iron to plants (Kraemer et al. 2006). Iron is one of the fourteen essential mineral elements for plants; it functions to accept and donate electrons and plays important roles in the electron transport chains of photosynthesis and respiration as well as involvement in chlorophyll synthesis. Other involvement includes nitrogen fixation and plant hormone synthesis (Connolly and Guerinot, 2002; Kim and Guerinot 2007; Briat 2008; Charlson and Shoemaker 2006; Hiradate et al. 2007). Iron is also one of three nutrients in addition to nitrogen and phosphorus that most commonly limit plant growth (Guerinot, 2001). Specifically for photosynthesis, iron is involved in electron transport through Photosystem II and Photosystem I and in coordinating the flow of electrons from Photosystem II to Photosystem I. The reaction center of photosystem II contains iron in the ionic form and that of photosystem I as three molecules of 4Fe-4S type iron sulfur clusters. The iron-sulfur clusters function as redox signals for light dark regulation of the Calvin cycle (Briate 2008; Shanna 2006). Photosystem I is affected by iron deficiency to a larger extent than Photosystem II probably because it contains a very large number of iron containing components (Sharma 2007). The synthesis and metallation of chloroplast electron transport components slows down as the electron transport system develops under the influence of iron deficiency and the seed stored iron is exhausted. Under conditions of iron deficiency, the expression of genes encoding chlorophyll-binding proteins would be downregulated, leading to the remodeling of the antenna components to adapt to the potentially photoinhibitory conditions resulting from reduced electron transport capacity (Sharma 2007). Iron is also forms a vital part of cytochromes, globins, various redox enzymes, and is a constituent of nitrogenase; it is a component in haem proteins such as cytochromes, catalase, and Fe-S proteins such as ferredoxin (Surridge 2001; Ma 2005; Epstein 1972; Curie and Briat 2003). In addition to 3 a decrease in the amount of chlorophylls from iron deficiency, there is also a decrease in the amount of carotenoids, but this decrease is not as pronounced ( Abadia, J. and Abadia, A. 1993 ). Iron exists in two valence states, ferric (Fe3J and ferrous (Fe2J. Ferrous iron is more easily taken up and used by plants than ferric iron, although under the usually prevailing conditions in soils and biological fluids, iron is predominantly in the ferric state. The soil pH strongly influences iron solubility and therefore availability to plants. Iron is insoluble at neutral or basic pH levels in the soil and the iron concentrations in solution decrease when the pH increases. Iron solubility is also influenced by several soil properties such as the redox conditions and the presence of organic matter (bacterial siderophores for example) (Briat 2008; Brown and Jolley 1989). Water and oxygen liberate iron by weathering primary minerals. The weathering of iron bearing primary minerals leads to formation of secondary iron oxides and the release of Fe2+ and Fe3+. In most soils, iron oxides and hydroxides are the largest pool of iron. The low solubility of iron oxides in the neutral pH range is responsible for low dissolved iron concentrations that induce iron deficiency symptoms in many plant species (Briat 2008; Kraemer et al. 2006). In the rhizosphere, plant-microorganism interactions can be described as a feedback loop during which (1) plant roots secrete exudates that modify the rhizospheric environment; (2) these changes are sensed by the resident microbial populations; (3) selection of adapted microbial population to the new environment occurs, leading to a modification of the structure and activities of the microbial community; (4) in turn, these variations will influence plant growth and physiology, leading to changes in plant root exudates, and so on. To be more specific, microorganisms found in the soil may also enhance iron uptake in plants (Briat 2008; Fodor 2006). The seed mineral content of iron depends on factors such as mobilization from the soil, uptake by the roots, movement within the plant, and finally iron deposit in the forming seeds (Chauhan 2006). Iron is toxic at high levels and because of this potential, cells store iron with the specialized iron storage protein, ferritin. While animal ferritins are found in the cytosol, plant ferritins contain transit peptides for delivery to organelles called plastids and whose abundance is strictly controlled at the 4 transcriptional level by the iron status of the cells (Connolly and Guerinot 2002; Briat 2008; Nilsen and Orcutt 1996; Suzuki et al. 2006; Gross et al. 2003). The difference between plant ferritin genes and animal ferretin is that plants do not contain Iron Regulatory Elements (found in animal ferritin genes), but instead have Iron-Dependent Regulatory Sequences (IDRS), responsible for the repression of transcription under low iron supply (Gross et al. 2003). In most plants (excluding grasses), iron deficiency is associated with morphological changes such as the inhibition of root elongation, increase in the diameter of apical root zones, and abundant root hair fonnation. This occurs due to the fonnation of transfer cells that are part of the mechanism to enhance iron uptake and the site of iron deficiency induced root responses in Strategy I plants (Marschner 1995). Iron Uptake Mechanisms of Plants Strategy I plants exude protons into the rhizophere as well as reductants/chelators such as electrons (e·), organic acids, and phenolics, lowering the pH of the soil solution and increasing the solubility ofFe3+. The release of protons is mediated via a P-type ATPase located in the plasma membrane. Ferric iron is reduced to Fe2+ at the surface of the plasma membrane of root epidennal cells by ferric chelate reductase (Alam et al. 2005; Brown 1978; Kim and Guerinot 2007; Brown and Jolley 1989; Ma 2005; Curie and Briat 2003; Guerinot and Yi 1994; Hiradate et al. 2007). Fe2+ is transported into the root by IRTI (iron regulated transporter 1) protein, a member of the ZIP metal transporter family (zinc and iron regulated transporter family), an inducible Fe2+ transporter that contributes to metal homeostasis (Colangelo and Guerinot 2006; Connolly and Guerinot 2002; Kim and Guerinot 2007; Ma 2005; Hiradate et al. 2007; Kraemer et al. 2006). Strategy I plants also show enhanced development of lateral roots and differentiation of specialized transfer cells. These changes increase the surface area for reduction and transport of iron (Guerinot and Yi 1994). The iron deficiency induced root response in Strategy I is located in the apical root zones of the plants (Marschner et al. 1987). Figure 1 shows the path of iron acquisition in Strategy I plants. 5 Fe(II) Cytoplasm H / 1\ Cell Membrane Soil Environment H '· . l -:::----1 Fe(III) Fe(II) Soluble Insoluble Figure 1: Strategy I uptake. This represents the proton release from a Strategy I plants where the insoluble iron is reduced to a soluble form and then adsorbed through the cell membrane of the root via IRTl. (Adapted from Ma 2005) Graminaceous species, such as com, wheat, oats, and rice, use the chelation-based Strategy II, which retrieves adequate amounts of iron from soils containing scant amounts of iron (Kim and Guerinot 2007; Ma 2005; Curie and Briat 2003). In response to iron deficiency, the grasses secrete Fe3+ chelators called phytosiderophores, which are small molecular weight compounds that retrieve insoluble iron and make it available to the plant (Alam et al. 2005; Connolly and Guerinot 2002; Kim and Guerinot 2007; Brown and Jolley 1989; Curie and Briat 2003; Takagi 1993). The complex is then absorbed into the plant via a species specific transporter. Figure 2 illustrates the Strategy II uptake mechanism. 6 Cytoplasm plant specific phytosiderophore 8 ~hytosiclerophore) Cell Membrane Soil Environment @ plant specific phytosiclerophore Fe(III) complex Figure 2: Strategy II uptake. This represents the chelation based strategy of grasses where each species puts out its own phytosiderophore which complexes with Fe3+ and then is transported back into the plant via a phytosiderophore-iron transporter. (Adapted from Ma 2005) Phytosiderophores Phytosiderophores were first observed in rice by Takagi in 1976 (Takagi 1976; Bughio et al 2002). The chelation strategy is more efficient than the reduction strategy and allows grasses to survive under more drastic iron deficiency conditions (Marschner et all 986). Charlson and Shoemaker (2006) stated that because dicots, gymnosperms, and monocots (both grass and non-grass species) show Strategy I physiology, and Strategy II is unique to grasses, it can be postulated that during evolution either grasses gained Strategy II or all other plant species lost Strategy II. They also saw conservation of the Strategy I genes among dicots, grasses, and a gymnosperm suggesting that Strategy I may have been established in gymnosperms upon the appearance of flowering plants (Charlson and Shoemaker 2006). Grasses can grow via Strategy II because it is less pH dependent than Strategy I (Guerinot and Yi 1994). Phytosiderophores bind tightly to the sparingly soluble iron in the soil, and are then reabsorbed into the roots, taking their chelated iron with them (Surridge 2001 ). The phytosiderophore system of iron uptake resembles the siderophore system attributed to microorganisms. The affinity phytosiderophores 7 for Fe3+ is two to three orders of magnitude higher than for synthetic iron chelates such as FeEDDHA while microbial sideophores such as ferrioxamine B have a higher affinity for iron.. One reason is that a natural chelator is more stable than a synthetic chelator (Alam et al. 2005; Bocanegra et al. 2004; Marschner et al 1987; Guerinot and Yi 1994; Marschner 1995). This strategy is clearly less sensitive to pH than the reduction strategy, but there is a cost to synthesizing and releasing phytosiderophores because they may or may not be recovered after release into the rhizosphere. However, there is a strong positive correlation between the amounts of phytosiderophores released from graminaceous plants and the resistance of those plants to iron deficiency (Guerinot 2001; Guerinot 2007; Takahashi et al. 2001). The process of iron acquisition by Strategy II plants can be characterized by the biosynthesis of phytosiderophores inside the roots, secretion of phytosiderophores into the rhizosphere, solubilization of insoluble iron in soils by chelation of phytosiderophores, and the uptake of phytosiderophore-Fe3+ complexes by the roots (Ma 2005; Murata et al. 2006; Hiradate et al. 2007; Ma and Nomoto 1996). The regulation of iron uptake in roots of iron deficient graminaceous species is induced at two levels increase in rates of release and uptake of the iron-phytosiderophores (Marschner et al. 1987). Thermodynamic and kinetic considerations are equally important in the understanding of strategy II iron acquisition based on subjects such as ligand affinity and the stability of complexes (Kraemer et al. 2006). The iron-efficiency of Strategy II genotypes is essentially a function of the capacity of plant roots to produce and release phytosiderophores in response of iron deficiency stress and how the phytosiderophores are taken up. The phytosiderophores released by the roots in response to iron deficiency form a complex with Fe3+ and contribute to increased uptake ofFe3+ in the chelated form. The capacity to produce and release the phytosiderophores is genetically determined. Those plants that show tolerance to iron chlorosis on calcareous soils have been found to be more efficient in release of phytosiderophores (Sharma 2006). Mugineic Acid Mugineic acid was first identified as a phytosiderophore in barley and its analogues have since been isolated and identified from various graminaceous species. All form water soluble 1: 1 complexes 8 with Fe3+ (Namba et al. 2007). Mugineic acid and its derivatives (i.e. other phytosiderophores) are hexadentate ligands with aminocarboxylate and hydroxycarboxylate functional groups. The ligands are synthesized by hydroxylation of nicotianamine from methionine (Kraemer et al. 2006). Iron deficiency stress causes the amount of MAs secreted to increase dramatically and the amount of MAs secreted from the roots of graminaceous plants determines the amount of tolerance to deficiency stress a plant has. For example, rice secretes small amounts of MAs and is susceptible to iron deficiency, whereas barley secretes relatively high levels of MAs and can tolerate iron deficiency (Hansen et al. 1996; Higuchi et al. 2001; Ishimaru et al. 2006; Guerinot 2007; Kim and Guerinot 2007). Wheat is also known to secrete larger amounts of phytosiderophores than maize (Reichard et al. 2005). MAs are synthesized from methionine and Met synthesis is associated with the Met cycle (Astolfi et al. 2003; Connolly and Guerinot 2002; Ma 2005). The Met cycle is highly active in roots, allowing for the increased demand of Met required for the synthesis ofMAs (Kobayashi et al. 2005). The family of mugineic acids includes MA, 2'-deoxymugineic acid (DMA), 3-epihydroxymugineic acid (epi-HMA), and 3-epihydroxy 2'-deoxymugineic acid (epi-HDMA) (Kim and Guerinot 2007; Curie and Briat 2003; Ma and Nomoto 1993; Kawai et al. 1993). In oats, avenic acid A is biosynthesized from 2'deoxymugineic acid by cleavage of the azetidine ring (Ma 2005; Ma and Nomoto 1993). 3-hydroxy-2'deoxymugineic acid (HDMA) from L. perenne, and 2 '-hydroxyavenic acid A (HAVA) from P. pratensis were the most recent phytosiderophores identified (Ueno et al. 2007). DMA has been shown to be secreted from two cultivars of F. rubra (Rubina and Bamica) as well (Ma et al. 2003). Each grass produces its own set of MA and production and secretion of MAs are increased in response to iron deficiency (Kim and Guerinot 2007; Curie and Briat 2003; Ma and Nomoto 1993; Kawai et al. 1993). The initial step in the production of MAs is the condensation of three molecules of S..adenosyl methionine (SAM) to produce one molecule of nicotianamine (NA) (Curie and Briat 2003; Negishi et al. 2002; Roje 2006). SAM is the substrate of a variety of other reactions in plants that lead to the biosyntehsis of ethylene, polyamines, nicotianamine, phytosiderophores, and biotin. SAM is the methyl group donor in a wide variety of enzyme catalyzed reactions in plants. 0-, N-, and C-methyltransferases 9 are the three major families of methyltransferases based on the chemical nature of the substrate. Some methyltransferases exhibit strict specificity for a single substrate; many others accept a broad range of substrates (Roje 2006). The general biosynthetic pathway of mugineic acid phytosiderophores begins with L-methionine as stated above. S-adenosyl methionine synthase converts L-methionine to S-adenosyl methionine. Nicotianamine synthase then converts the molecule to nicotianamine. Nicotianamine is converted to 3 'oxo intermediate by nicotianamine aminotransferase. Deoxymugineic acid synthase converts the intermediate to 2'-deoxymugineic acid (OMA). From here, dioxygenases change OMA into other phytosiderophores such as 3'-epihydroxy mugineic acid and 3'-epihydroxy-2-deoxymugineic acid (Callahan et al. 2006; Sharma 2006; Suzuki et al. 2006; Bashir et al. 2006). Deoxymugineic acid synthase (OMAS) is responsible for the synthesis of OMA by reduction of a 3"-keto intermediate. DMAS was identified as a putative member of the aldo-keto reductase superfamily, upregulated under iron deficient conditions as was studied in barley and rice (Bashir et al.2006; Briat 2008). The synthesis of mugineic acids (MA) from methionine also induces the synthesis of ethylene in iron deficient plants. Ethylene is a by-product in the MA synthesis pathway (Nilsen and Orcutt 1996). Grasses are the only species that have the ability to convert nicotianamine (NA) to MA by way of the enzyme nicotianamine aminotransferase (NAAT). NAAT activity is dramatically induced by iron deficiency and suppressed by iron resupply (Briat 2008). Other genes involved in the biosynthesis of phytosiderophores includes APRT (adeninephosphoribosyl transferase ), SAMS (S-adenosy }methionine synthetase), NAS (NA synthase), NAA T (NA aminotrasferase), and iron deficiency specific clones IDS2 and IDS3 (Murata et al. 2006). Methionine recycling for the production of MAs occurs with the help of the Yang cycle (Negishi et al. 2002). Iron deficiency tolerances depends on an increase in the activity of both NAS and NAAT, which are also crucial for the enhanced secretion of MAs (Higushi et al. 200 I). Higuchi et al (2001) showed that the concentration of NA in rice leaves is rather high and that rice NAS synthase was induced under iron deficiency not only in roots but also in the leaves. Their results suggest that rice NAS may have roles other than just the biosynthesis of MAs, such as transport 10 within the plant itself (Higuchi et al. 2001; Takahashi et al. 2001). Nicotianamine also appears to be required for plant fertility. Flowers in iron deficient plants are abnormally shaped and pollen count is usually lower. NA may function in long distance transport of iron to the reproductive organs and in other cases as a scavenger of Fe2+ in order to prevent oxidative stress to the plant cells (Briat 2008; Callahan et al. 2006). Nicotianamine synthase is a key enzyme in MA biosynthesis, catalyzing three molecules of SAM into one molecule of nicotianamine. NAS activity in graminaceous plants is well correlated with tolerance to iron deficiency (Mizuno et al. 2003). NA has been shown to chelate Fe2+, in particular at a higher pH, which may allow it to serve as an intracellular Fe2+ scavenger thereby protecting the cell from Fe2+-mediated oxidative damage (Schaaf et al. 2004). More specifically, the synthesis of nicotianamine from three molecules of SAM includes two carboxypropyl group transfers and one azetidine ring formation, with three molecules of 5 ' rnethylthioadenosine (5'-MTA) released. All three reactions are catalyzed by the enzyme nicotianamine synthase. In non-graminaceous plants, nicotianamine is thought to bind metal ions and to participate in their trafficking inside the plant. In graminaceous plants, nicotianamine is also the precursor of phytosiderophores, which are essential in acquiring iron from soil (Roje 2006; Romhelf and Schaaf 2004; Bashir et al. 2006; Higuchi et al. 1999). Nicotianamine synthase (NAS) catalyzes the bonding of three Sadenosyl Met molecules to form nicotianarnine (NA), which is then converted to deoxymugineic acid by an amino group transfer catalyzed by nicotianamine aminotransferase (NAA n and subsequent reduction at the 3 '-carbon of the keto acid (Bashir et al. 2006; Takahashi et al. 1999). Phytosiderophore Secretion Phytosiderophore secretion is known to follow a diurnal rhythm, being biosynthesized during the day and accumulating within the roots until secretion the following morning (Connolly and Guerinot 2002; Ma 2005; Ueno et al. 2007; Reichman and Parker 2007). The trigger for phytosiderophore release may be a response to some historical rather than current light conditions (Reichman and Parker 2007). The amount ofbiosynthesis is dependent on the extent ofiron deficiency (Ma and Nomoto 1996). 11 Generally, plants start to secrete phytosiderophores every morning from the third hour after the sunrise or the onset of the light period. The secretion continues for 3 hours. The timing of secretion is controlled by the temperature around the root environment; when the temperature is high, the secretion occurs early, while secretion is delayed when the temperature becomes low (Ma 2005; Ma and Nomoto 1996; Reichard et al. 2005). The diurnal rhythm of phytosiderophore secretion would be advantageous for iron acquisition because more concentrated phytosiderophores could be released into the rhizosphere and microorganisms that degrade phytosiderophores could not follow the elongation of root tips, thereby phytosiderophores could persist longer and acquire more iron (Hiradate et al. 2007). The diurnal release of phytosiderophores creates a strong nonsteady state with respect to the solution saturation state and surface chemistry of the different iron oxides. These conditions then trigger fast iron oxide dissolution reactions if other ligands, such as oxalate, citrate, or malate, are also present thereby releasing the iron to complex with phytosiderophores (Kraemer et al. 2006). The apical root zones are the sites of phytosiderophore release (Marschner et al. 1987). Under iron deficiency, plants can increase phytosiderophore release by about 20 times. At the same time, the uptake rate increases by only about 5 times. This finding suggests that the mechanisms for release and uptake of phytosiderophores are under separate genetic control (Dakora and Phillips 2002). In addition to phytosiderophores, Strategy II plants release other organic ligands as well. This process could be a relic of any Strategy I mechanisms that may have remained with the plants during evolution (Reichard et al. 2005). Following secretion, phytosiderophores solubilize soil iron by chelation, forming a complex with iron at a ratio of 1: 1 (Ma 2005). Iron solubilization depends on chelation ability and affinity (Ma and Nomoto 1996). Phytosiderophores can scavenge iron from a range of iron bearing minerals and soluble iron species, which are the ultimate sources of iron in soils (Kraemer et al. 2006). There is a change in the shape of the vesicles in root cells ofiron deficient plats that corresponds to the diurnal secretion ofphytosiderophores. The vesicles stay swollen until the onset of phytosiderophore secretion, and become shrunken by the end of secretion. These particular vesicles are 12 the sites of phytosiderophore synthesis and accumulate in the epidermal cells at the cell periphery facing the rhizosphere just before sunrise (Negishi et al. 2002; Higuchi et al. 1999). These vesicles were found to be covered with ribosomes and are thought to originate from the rough ER {Takahashi et al. 1999). There is also an increase in the root hairs under iron deficiency conditions that could results in increased root surface area and increased contact with soil iron. The conclusion was that this response is probably advantageous to rapidly increase phytosiderophore release, since it was evident that the release and biosynthesis of phytosiderophores are mainly localized in the apical root zone (Zhang et al. 2003). Yellow Stripe 1 Fe3+-MA complexes are transported into the plant via specific plasma membrane transporters located in the root (Connolly and Guerinot 2002; Kim and Guerinot 2007). Maize is the model grass organism that has been used to characterize the Fe3+-MA transport system (Briat 2008). The yellow stripe I (ysl) maize mutant showed a defect in uptake of iron-phytosiderophore complexes, resulting in iron deficiency and plants developing interveinal chlorosis (yellow-stripe) (Kim and Guerinot 2007). Yellow stripe I was first identified in 1929 by George W. Beadle, through its distinctive phenotype in which iron deficiency leads to incomplete pigmentation of leaves showing up as alternating yellow and green stripes running the length (Surridge 2001 ). Curie et al (2001) found that YSl cDNA has the ability to allow growth of iron-deficient yeast mutants on Fe-DMA in the presence ofBPDS, an iron-chelator, strongly suggesting that YSl is a transporter of phytosiderophore-bound Fe3+ (Curie et al. 2001). YSJ encodes an iron phytosiderophore transporter, an integral membrane protein with 12 putative transmembrane domains that belongs to the oligopeptide transporter ( OPT) superfamily (Kim and Guerinot 2007; Surridge 2001; Briat 2008; Curie and Briat 2003). The OPT family transports tri-, tetra-, penta-, and hexapeptides. It has also been noted that since phytosiderophores and peptides are amino acid derivatives, and since all functionally characterized members of the family function with inwardly directed polarity, all members of the OPT family may function in the uptake of amino acid containing compounds and their derivatives (i.e. 13 phytosiderophores) (Yen et al. 2001 ). The gene also has an E rich N-terminal region containing a specific motif (REG LE) known for its interaction with Fe3+ (Briat 2008). In barley, the HvYSJ gene was cloned and the protein it encodes was shown to have the same characteristics as Zm YSI, although it seemed to have a metal specificity restricted to Fe3+. In addition, in situ hybridization analysis of iron deficient barley roots revealed that the HvYSl mRNA was localized in epidermal root cells, as well as the protein as shown by immunohistological staining with an antiHvYSl polyclonal antibody (Briat 2008). HvYSI also belongs to the oligopeptide transporter (OPT) family, which transports oligopeptides in sever&l organisms, including bacteria, archaea, fungi, and plants. Hv YS 1 is the closest homo log to Zm YS 1 with 72. 7% identity and 95 .0% similarity (Murata et al. 2006). The YS 1 transporter from maize also showed a broad specificity as it can transport various phytosiderophore-bound metals such as zinc, copper, and nickel, and nicotianamine complexed with nickel, ferrous (Fe2+) and ferric (Fe3 +) iron (Hiradate et al. 2007; Ma 2005; Murata et al. 2006). In spite of low sequence similarity, Murata (2006) found that the N-terminal domains of both Hv YS I and Zm YS I share the basic feature of being rich in glutamic and aspartic acid residues. Both proteins also have in this region an EXXE sequence, which is a F e3+ binding motif in a signal transduction system that responds to extracellular iron (Murata et al. 2006). Rice, however, has the ability to transport ferric iron by the phytosiderophore-mediated Fe3+ mechanism, but also possesses an Fe2+ uptake system with the use of a root specific IRTl-like F e2+ transporter (Bughio et al. 2002). This system is unique because rice has adapted for growth under submerged conditions, where Fe2+ is more abundant than Fe3+. Therefore, it is conceivable that Fe2+ is taken up by rice in a similar fashion to non-graminaceous plants. This strategy might have evolved specifically in rice plants because of the need to survive under submerged conditions, and growing well under Fe2+-rich conditions, which are encountered in paddy fields (lshimaru et al. 2006; Kim and Guerinot 2007). To address whether YS 1 might be involved in uptake of metal micronutrients other than iron, Roberts et al analyzed YS 1 gene and protein accumulation under conditions of metal deficiency. They 14 grew plants in modified Hoagland solution lacking iron, zinc, and copper. They found that Ysl expression failed to increase under conditions of copper or zinc limitation and that this may be taken as suggesting that YS 1 is not involved in the primary acquisition of these metals. It was also stated that although YSI does not transport zinc phytosiderophore complexes, another yellow-stripe-like transporter could be involved instead (Roberts et al. 2004). Schaaf et al also observed in three different systems that Zm YSI-mediated Fe3+-DMA transport depends on the availability of protons. They concluded that ZmYSl represents an W-Fe3+phytosiderophore cotransporter. Their investigation also uncovered another factor that contributes to more efficient iron acquisition in grasses: coupling the transport ofiron-phytosiderophores, which are negatively charged at neutral pH, to protons allows efficient root uptake even at high soil pH, because transport of the proton-coupled substrate can be driven as long as the overall difference of the electrochemical potential remains negative (Schaaf et al. 2004 ). Other metals thought to be transported by ZmYSl include Cd and Mn (Hill et al. 2002). Growth complementation and oocyte transport studies clearly showed that Zm YSI also transports phytosiderophore-chelated Zn2+. Zm YS 1 also transported phytosiderophore-chelated nickel and copper at a similar rate to iron and zinc (Schaaf et al. 2004). Barley was also investigated by Suzuki et al (2006) in the phytosiderophore uptake of zinc. However, the assays used were indirect methods to measure the amount of phytosiderophores and cannot distinguish mugineic acids from other metal chelating substances. This could be one reason why there is conflicting data as to whether or not phytosiderophore secretion increases under conditions of low zinc supply (Suzuki et al. 2006). The sequence homology ofHvYSl and ZmYSl in the N-tenninal outer membrane regions and the loops between the sixth and seventh transmembrane helices are significantly lower than the outer regions. They also showed that residues 314-385 in Hv YS 1 are responsible for the higher selectivity of Fe3+-DMA over Fe2+-NA. The suggestion by the authors was that the variable regions in the middle outer membrane loops are essential and sufficient to define the transport specificity, whereas the N-tenninal regions have no influence on the substrate specificity (Harada et al. 2007). 15 The variety of paralogs found within each family of transporters could be explained by at least three reasons: 1) affinity to a specific minerall; 2) placement in organs or tissues; 3) placement in subcellular compartments, such as vacuoles or plastids. Di:fferent iron carrier molecules may also explain the diversity of iron transporter families in plants. The YSl protein, for example, transporters Fe3+ phytosiderophores, and possibly Fe2+-NA, structurally similar to phytosiderophores. Yellow stripe like proteins are believed to mediate the uptake of metals that are complexed with nicotianamine and assist in the long distance transport of metals within the plant. Other metals than iron may rely on yellow stripe like proteins for transport into or within the plant itself (Chang et al. 1999; Colangelo and Guerinot 2006; Gross et al. 2003). One mechanism that is not understood is how plants sense iron and begin the processes of retrieving iron from the soil. Once these components are identified, the iron deficiency response will be better understood at the molecular level (Kim and Guerinot 2007). The purpose of this project was to identify and characterize the iron phytosiderophore transporter in Avena sativa. This species was selected because it produces a known phytosiderophore, avenic acid. Upon isolation, the transporter was compared to Hv YS 1 and Zm YS 1 to determine similarity between the sequences. The results from this project will enhance the understanding of iron-phytosiderophore mediated transport among graminaceous species as this information is currently limited. 16 Experimental Procedure: PiantGmwth: Seedlings were grown in Hoagland's medium for approximately 10 days. Plants were then transferred to distilled water (Figure 3) and grown until the appearance of yellowing on the leaves. Roots (Figure 4) were harvested and flash frozen in liquid nitrogen. Harvested roots were stored at -80°C. Figure 3: Oats grown hydroponically under iron deficient conditions. Figure 4: Oat roots in iron deficient hydroponic solution. RNA Isolation and c/gfilJJ!J2_: RNA was isolated from ground, frozen oat root tissue using the PureLink™ Plant RNA Reagent from Invitrogen. The reagent was added to ground, frozen plant tissue in order to lyse the cells. The 17 lysate was clarified by centrifugation. Addition of NaCl and chloroform separates the phases and RNA is then precipitated with isopropyl alcohol. RNA samples were then cleaned by DNase and RNeasy treatments. Amplification Grade Dexoyribonuclease I from Sigma was used to digest double and single stranded DNA. However, to be completely sure that the samples were free of contaminating DNA, the RNeasy protocol from Quiagen was used as well. This kit utilizes a silica membrane that also removes DNA from RNA samples; it will also remove the DNase that was used prior to the procedure. The integrity of the RNA sample was verified by gel electrophoresis. First Strand Synthesis and Amplification o{Specificity Region: RNA was used in the Transcriptor First Strand cDNA Synthesis Kit by Roche to obtain cDNA. A 20ul amplification using TITANIUM™ Taq PCR Kit from Clontech and the following primers was performed: Specificity region left: 5' -TTCGGTYTGAAGGCCTGGAAGCAGAC- 3' Specificity region right: 5' -CCATGTTGATGTCGGTGARCCC- 3' Gel verification provided visualization of the proper size fragment. Ligation into Vector and Cloning ofSpecificity Region· The TOPO TA Cloning® Kit for Sequencing by Invitrogen was used for sequencing preparations. The PCR product was ligated to the TOPO® Vector and allowed to incubate for 5 minutes at room temperature. One Shot® TOPl0 competent E.coli cells were used for chemical transformation. The cloning reaction was added to cells that were transformed following protocol and then plated onto LB plates containing 50ug/ml of ampicillin. Cells were grown overnight and white colonies were re-streaked onto the same type of LB plates and allowed to grow overnight. PCR verification of insert using the same protocol for the amplification of the specificity region. Colonies that showed insert were allowed to grow overnight in LB broth containing ampicilllin. Plasmid Isolation and Sequencing: 18 The QIAprep Spin kit from Qiagen was used to lyse bacterial cells, retrieve DNA, and prepared it for sequencing. Sequencing was performed by GENEWIZ, Inc., located in South Plainfield, NJ. 5' and 3' RACE The specificity region was located approximately in the middle of the gene; therefore to obtain the ends of the gene, 5' and 3' Rapid Amplification of cDNA Ends (RACE) was employed with the use of the GeneRacer™ Kit from Invitrogen. RACE amplifies full length 5' and 3' ends of cDNA using primers that were designed based on the sequence from HvYSl. Total RNA is dephosphorylated, decapped, ligated to an RNA Oligo which provides a known priming site, reverse transcribed with Superscript™ III RT and amplified using the following primers: Nested Primer: 5' - CGACACTGACATGGACTGAAGGAGTA - 3' GSP2' Primer: 5' -CTGTACGCGATCCAAGCGGGGAGAT- 3' It should be noted that between each step is an ethanol precipitation of the RNA. This step removes excess salts from the sample that could be problematic in later reactions. PCR Products were then cloned using the TOPO TA cloning kit mentioned above. The sequences were sent to GENEWIZ for sequencing. 3' RACE was performed by cDNA amplification using the RT template obtained from the reverse transcribing step of the protocol. Amplification was done using the GeneRacer™ 3' Primer and 3' RACE Forward Primer: GeneRacer 3' Primer: 5' - GCTGTCAACGATACGCTACGTAACG - 3' 3' RACE Primer: 5' - CGTGCTGAGCGTCGTTGCAGTGGT - 3' PCR products were cloned into cells using the TOPO TA Cloning Kit from Invitrogen, protocol listed above, and positive clones were sent to GENEWIZ for sequencing. Agrostis stolonifera specificity region: 19 To check the diversity in the specificity region, Agrostis stolonifera (creeping bentgrass) was used for a large scale RNA isolation followed by first strand synthesis and amplification using the specificity primers listed above. No further analysis was perfonned on this species. Primer Design All primers not provided in kits were designed by analyzing HvYSl and its homologs. In addition to HvYSl, ESTs from putative phytosiderophores from other grasses were also used in primer design. Table I lists all primers used in the sequencing of the putative iron-phytosiderophore transporter from A. sativa. Table 1: Primers. Primers used to amplify the sequence of the iron-phytosiderophore transporter in A. sativa. Primer Name Sequence Amplification Area Specificity Left 5' -TTCGGTYTGAAGGCCTGGAAGCAGAC - 3' Specificity region Specificity Right 5' -CCATGTTGATGTCGGTGARCCC-3' Specificity region GeneRacer 3' 5' - GCTGTCAACGATACGCTACGTAACG-3' 3'end Avena Sativa 3' End 5' -CGTGCTGAGCGTCGTTGCAGTGGT- 3' 3'end GeneRacer 5' 5' - CGACACTGACATGGACTGAAGGAGTA- 3' 5'end 5' - CTGTACGCGATCCAAGCGGGGAGAT- 3' 5'end Primer Nested Primer GSP2' Notes: All PCR products were purified using the QIAquick Gel Extraction Kit. 20 Res_u/ts;_ Harada et al (2007) has shown that there is an area in HvYSl that confers higher specificity for iron over the non-iron specific phytosiderophore transporter Zm YSl. This prompted the questioning of a specificity region within the Avena sativa iron-phytosiderophore transporter. The DNA sequence that was first isolated from Avena sativa was the putative specificity region utilizing the Specificity Primers listed in Table 1. A 533 base pair sequence was retrieved. The pink colored region in Figure 10 represents the specificity region of the gene. Nucleotide BLAST analysis yielded the best match as "Hordeum vulgare HvYSl mRNA for iron-phytosiderophore transporter, complete eds", accession number AB214183. There was an 83% identity between the sequences. Residues 793-1324 in Figure 11 represent this same region within the full gene BLAST analysis. When translated, the product is 177 amino acids in length. The gold area in Figure 12 represents the specificity region within the amino acid sequence. A protein BLAST reinforced the results of the nucleotide BLAST; residues 265-441 of Figure 13 are the specificity area, reinforced by the pink area to further show the diversity. A ClustalW2 analysis, Figure 14, further highlights the diversity in the specificity region, shown by the area in green. In this type of analysis, the"*" represents an identical amino acid between the two sequences,":" represents a conserved amino acid substitution, and"." represents a semi-conserved substitution. If none of those three symbols is shown beneath the alignment, then the substitution shows no conservation between sequences. Residues 265-441 of the ClustalW2 output represent the sequenced specificity region found in A. sativa. The putative specificity region also contained a conserved domain, the Oligopeptide Transporter (OPT) domain. This is expected because the gene is a membrane transporter found in the roots of A. sativa. This result is published on GenBank under EU619717 and ACC77193 The specificity region of Agrostis stolonifera was also identified using the same primers that were involved in the identification of the specificity region in A. sativa. Figure 5 shows the nucleotide sequence result from Gene Wiz. The nucleotide BLAST in Figure 6 shows that the best match was the published specificity region of A. sativa while Figure 7 shows the next best match as HvYSl. The protein sequence is 171 amino acids in length and the highlighted area in Figure 8 shows A. stolonifera diversity area. 21 GGCCTGGAAGCAGAC ATTCTTCTTIGACTTTAGCATGACATATGTCGGTGCCGGGATGATITGCCC GCATATAGTAAATATCTCCACCCTCCTTGGCGCAATTCTITCATACGGATTATTGTGGCCACTCATCAG TAAGAACAAGGGGGATTGGTACCCTGCAAATGTACCAGAAAGCAGCATGAAAAGTTIGTACGGTTAC AAGGCCTTCATATGTATCGCTCTGATCATGGGGGATGGACTCTACCACTTCACAAAAATTATTAGCAT CACTTTIAAGGGCATGTATCGACAGTTIAGCCGTAAGCGCGTTGACAATCGAGTGAAAAATGTGGAC AATACGGTCTCACTTGAGGATCTGCAGCGCGATGAGATCTTCGGAAAGGGCCATATCCCAGCTTGGAT GGCTTACACTGGGTATGCCGTGTTAAGCGTCGTTGCGGTGGTTACCACGCCAATAATGTTIAGACAGG TGAAATGGTACTACGTAGTTATAGCCTATATGGTCGCCCCCGTTCTTGGATTCGCCAATTCCTATGGG ACAGGGCTCACCGACATCAACATGG Figure S. Specificity Region of Agrostis stolonifera. The specificity region of A. stolonifera was sequences using primers Specificity Left and Specificity Right. Primers are shown in red. Avena sativa putative iron-phytosiderophore transporter mRNA, partial eds Lengths=755 Score= 669 bits (362), Expect= 0.0, Identities= 471/523 (90%), Gaps= 10/523 (1%) Strand=Plus/Plus Agros 1 ATTCTTCTTTGACTTTAGCATGACATATGTCGGTGCCGGGATGATTTGCCCGCATATAGT 60 I II I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I Avena 238 ATTCTTCTTTGACTTTAGCCTGACATATGTCGGTGCCGGGATGATTTGCCCACATATAGT 297 Agros 61 AAATATCTCCACCCTCCTTGGCGCAATTCTTTCATACGG-ATTATTGTGGCCACTCATCA 119 111111 11111111 11111 11111 11111 11 11 11 11111111111111111 Avena 298 AAATATATCCACCCTTCTTGGTGCAATCCTTTCTTATGGGAT-ATTGTGGCCACTCATCA 356 Agros 120 GTAAGAACAAGGGGGATTGGTACCCTGCAAATGTACCA-GAAAGCAGCATGAAAAGTTTG 178 11111111 I 1111 1111111111I111111111 I I 111111111111111111 11 Avena 357 GTAAGAACAAGGGTGATTGGTACCCTGCAAATGT-CAAAGAAAGCAGCATGAAAAGTCTG 415 Agros 179 TACGGTTACAAGGCCTTCATATGTATCGCTCTGATCATGGGGGATGGACTCTACCACTTC 238 11 I I I I I I I I I I I I I 11111111111111111111111111111111111111111111 Avena 416 TATGGTTACAAGGCCTTCATATGTATCGCTCTGATCATGGGGGATGGACTCTACCACTTC 475 Agros 239 ACAAAAATTATTAGCATCACTTTTAAGGGCATGTATCGACAGTTTAGC-CGTAAGCGCGT 297 Avena 476 ACCAAAATTATTACCATCACTTTCAAGGGCATGTATCGACAGTTTA-CTCGTAAACGTGC 534 Agros 298 TGACAATCGAGTGAAAAATGTGGACAATACGGTCTCACTTGAGGATCTGCAGCGCGATGA 357 11 1111111111 111111 1111 111111111 I I I I I l 1111111111111111 111111111111111 I I I I I I I I I 11 I 11111 11 I 111111111111111 I I I I Avena 535 TGACAACCGAGAGAAAAATGTGGACAATACGGTCTCGCTCGAGGATCTGCAGCGCGACGA 594 Agros 358 GATCTTCG-GAAAGGGCCATATCCCAGCTTGGATGGCTTACACTGGGTATGCCGTGTTAA 416 I IIIII I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I I I l I I I Avena 595 GGTCTTCAAGAA-GGGCCATATCCCCGCTTGGATGGCGTACAGTGGGTACGCCGTGTTGA 653 Agros 417 GCGTCGTTGCGGTGGTTACCACGCCAATAATGTTTAGACAGGTGAAATGGTACTACGTAG 476 I I I I I I I I I I I l I I I I I I I I I I I II I I I I I I IIII IIIIIIIIIIIIII II I Avena 654 GCGTCGTTGCAGTGGTGACCACGCCTATAATGTTCCGACAAGTGAAATGGTACTATGTTG Agros 477 TTATAGCCTATATGGTCGCCCCCGTT-CTTGGATTCGCCAATT I I I I I 1 I II I I I Avena 714 713 518 I I I I I I I I I I I I I I I I I I I I II I I I TTATAGCCTATGTCATCGCCCC-GATGCTTGGATTCGCCAATT 755 Figure 6. Nucleotide BLAST of A. stolonifera specificity region. Nucleotide BLAST results against A. sativa specificity region. 22 Hordeum vulgare HvYSl mRNA for iron-phytosiderophore transporter, complete eds Length=2430 Score= 508 bits (275), Expect (2%) Strand=Plus/Plus Agros 1 8e-141, Identities= 453/539 (84%), Gaps= 12/539 ATTCTTCTTTGACTTTAGCATGACATATGTCGGTGCCGGGATGATTTGCCCGCATATAGT 60 I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I l I I I I I I I I HvYSl 966 ATTCTACTTTGACTTTAGCATGACATACGTTGGTGCCGGGATGATTTGTCCACATATAGT 1025 Agros 61 AAATATCTCCACCCTCCTTGGCGCAATTCTTTCATACGGATTATTGTGGCCACTCATCAG 120 111111 11 I I I l 1111111 111111 I 1111 111 11 1111111111111111 HvYSl 1026 AAATATATCTACCCTCCTTGGTGCAATTATCTCATGGGGAATAATGTGGCCACTCATCAG 1085 Agros 121 TAAGAACAAGGGGGATTGGTACCCTGCAAATGTACCAGAAAGCAGCATGAAAAGTTTGTA 180 II I 11 11 11 I I I I I I 11 I I I I I I I I I I I I I 11 I 111 I I I I I I I I I I I I I I 11 I I I I I I HvYSl 1086 TAAAAACAAGGGGGATTGGTACCCTGCAAAAGTACCAGAAAGCAGCATGAAAAGTTTGTA 1145 Agros 181 CGGTTACAAGGCCTTCATATGTATCGCTCTGATCATGGGGGATGG-ACTCTACCACTTCA 239 111111111111111111111 II 11111 11111111111111 I I 1111111111 HvYSl 1146 CGGTTACAAGGCCTTCATATGCATAGCTCTCATCATGGGGGATGGCA-TGTACCACTTCA 1204 Agros 240 CAAAAATTATTAGCATCACTTTTAAGGGCATGTATCGACAGTTTAGCCGTAAGC-GCGTT 298 HvYSl 1205 TAAAAATTGTTGGCATCACTGCTATGAGCATGTATCGGCAATTTAGCCACAAGCAG-GTT 1263 Agros 299 GACAATCGA-GTGAAAAATGTGGACAATACGGTCTCACTTGAGGA-TCTGCAGCGCGATG 356 1111111 II 11111111 IIII I I I II I 1111111111 II 1111111 1111 I Ill I I I I II I I I I I I I I I I I I I 11 I I 111 I I I I I I 11 I I HvYSl 1264 AACAA-CAAAGCAAAAAATGCGGACGACACTGTCTCGCTTGAGGAGT-TACACCGCCAGG 1321 Agros 357 AGATCTTCG-GAAAGGGCCATATCCCAGCTTGGATGGCTTACACTGGGTATGCCGTGTTA 415 1111111 II Ill Ill 11111 1111111111 Ill 1111 111111 1111 HvYSl 1322 AGATCTTTAAGAGAGG-CCACATCCCCTCTTGGATGGCATACGCTGGTTATGCCTTGTTT 1380 Agros 416 AGCGT-CGTTGCGGTGGTTACCACGCCAATAATGTTTAGACAGGTGAAATGGTACTACGT 474 11111 I 1111 11111111 I Ill 1111111 I Ill 11111 11111111 II HvYSl 1381 AGCGTTC-TTGCAGTGGTTACAATACCAGTAATGTTCAAACAAGTGAAGTGGTACTATGT Agros 475 AGTTATAGCCTATATGGTCGCCCCCGTTCTTGGATTCGCCAATTCCTATGGGACAGGGC 111111111111 HvYSl 1440 I II 111111 1439 533 I 11111111 11111111 II 1111111111 TGTTATAGCCTATGTCGTTGCCCCCATGCTTGGATTTGCCAATTCATACGGGACAGGGC 1498 Figure 7. Nucleotide BLAST of A. stolonifera specificity region. Nucleotjde BLAST results of A. stolonifera specificity region against HvYSI. MTYVGAGMICPHIVNISTLLGAILSYGLL WPLISKNKGDWYPANVPESSMKSL YGYKAFICIALIMGDGLY HFTKilSIT AVVTTPI MFRQVKWYYVVIAYMVAPVLGFANSYGTG Figure 8: Amino Acid sequence of A. stolonifera specificity region. The highlighted area shows the diversity region of A. stolonifera specificity region. The sequence is 171 amino acids in length. 23 Another Clustal2W analysis was run on the specificity regions obtained from both A. sativa and A. sto/onifera against the published protein sequences of Hv YS 1 and Zm YS 1. Figure 9 shows the results of this analysis with the area of diversity once again highlighted. There is also a point in Zm YS 1 where it has a glycine and no other sequences contain that particular amino acid, this is highlighted in green. It is a small amino acid, so it may not change the 3-D structure of the protein very much. The sizes of the amino acids may play an important role in the specificity of the transporter since the structure is altered. A future analysis on the protein crystal structure would aid in determining why ZmYSl takes up additional metal-phytosiderophore chelates whereas HvYSl only accepts iron-phytosiderophore chelates. A similar analysis would benefit A. sativa in determining the iron specificity of the transporter. Agrostis Avena HvYSl ZmYSl -----------------------------------MTYVGAGMICPHIVNISTLLGAILS -----------------------------FFFDFSLTYIGAGMICPHIVNISTLLGAILS SFFQWFYTGGDACGFVQFPTFGLKAWKQTFYFDFSMTYVGAGMICPHIVNISTLLGAIIS SFFQWFYTGGEVCGFVQFPTFGLKAWKQTFFFDFSLTYVGAGMICSHLVNISTLLGAILS 25 296 297 300 :**:******.*:**********:* Agrostis Avena HvYSl ZmYSl YGLLWPLISKNKGDWYPANVPESSMKSLYGYKAFICIALIMGDGLYH YGILWPLISKNKGDWYPADVKESSMKSLYGYKAFICIALIMGDGLYH WGIMWPLISKNKGDWYPAKVPESSMKSLYGYKAFICIALIMGDGMYH WGILWPLISKQKGEWYPANIPESSMKSLYGYKAFLCIALIMGDGTYH 85 356 357 360 :*::******:**:****.: *************:********* ** Agrostis Avena HvYSl ZmYSl AVVTTPIMF AVVTTPIMF AVVTIPVMF SAVIIPHMF • * Agrostis Avena HvYSl ZmYSl 144 415 416 420 * ** RQVKWYYVVIAYMVAPVLGFANSYGTG--------------------------------RQVKWYYVVIAYVVAPMLGFANSYGTG--------------------------------KQVKWYYVVIAYVVAPMLGFANSYGTGLTDINMGYNYGKIALFVFAGWAGKENGVIAGLV RQVKWYYVIVAYVLAPLLGFANSYGTGLTDINMAYNYGKIALFIFAAWAGRDNGVIAGLA 171 442 476 480 :*******::**::**:********** Figure 9: Clustal Alignment of specificity regions and known transporters. Clustal2W analysis of specificity regions of putative iron-phytosiderophore transporter in A. sativa, A. stolonifera, and published phytosiderophore transporters HvYS 1 and ZmYS 1. Diversity region highlighted in gold. The GeneRacer kit from Invitrogen was used to obtain the 5' and 3' ends of the gene using primers designed to flank the specificity region that was obtained first. Using primers Avena 3' Primer and GeneRacer 3' Primer listed in Table 1, the 3' end of the gene was isolated. An 808bp nucleotide sequence that corresponds to the blue highlighted region in Figure 10 was sequenced. The best match was also "Hordeum vulgare HvYSl mRNA 24 for iron~phytosiderophore transporter, complete eds", accession number AB214183. An 86% identity between the sequences was listed on the BLAST results. Nucleotides 1231-2024 in Figure 11 represent the 3' end of the putative iron-phytosiderophore transporter in A. sativa. The protein is a stretch of 268 amino acids shown in Figure 11 as a non-highlighted region towards the end of the gene. The area in purple represents an overlap between the specificity region and the 3' end of the gene. The protein BLAST also resulted in HvYSl as the best match with 88% identity between the sequences. Residues 409675 in Figure 12 represent the 3' end of the gene. Results of the protein BLAST were the conserved domains COG1297 superfamily and the OPT binding domain. The OPT domain was also a result in the bioinformatics of the specificity region. The COG1297 superfamily is listed as a predicted membrane protein with an unknown function. This fact that the sequence is falling into this category is not surprising because the ironphytosiderophore transporter is located in the membrane of root cells of graminaceous species. The Clustal2W analysis shows conservation between the putative iron-phytosiderophore transporter in Avena and HvYSl as well as ZmYSl. This can be seen in Figure 14, residues 360-678. As stated previously, there was an overlap between the sequences of the specificity region and 3' end of the Avena gene. This overlap was approximately 100 bases and is shown in gold in Figure 10 and in purple in Figure 12 where the combined pieces gave a protein sequence that was 412 amino acids in length. The combined sequences are 1,240 bases in length, approximately two-thirds of the gene. The final portion that needed to be identified was the 5' end of the gene. The first set of sequences produced a domain that was a methyltransferase and members of this family are SAM dependent methyltransferases. This is not surprising because SAM is in the pathway that produces phytosiderophores. Another domain was the dimerisation domain that is found at the N-terminus of a variety of plant 0methyltransferases. Although this is not the 5' end of the gene, it is something that would be found in the biosynthetic pathway of phytosiderophores. To isolate the 5' end of the gene, the 5' RACE Nested Primer and the GSP2' primer were used. A nucleotide sequence approximately 800bp in length was obtained. Figure 10 shows this area in maroon and the start codon is highlighted in turquoise. BLAST analysis came back with "Hordeum vulgare HvYSl mRNA for 25 iron-phytosiderophore transporter, complete eds", accession number AB214183 as the best match again. The identity here was at 88%. Figure 11 shows the 5' area as nucleotides 1-800. The protein sequence is 268 amino acids in length and the protein BLAST again revealed the presence of COG1297 and OPT domains. The pink area in Figure 12 represents the amino acids of the 5' end of the gene as well as the residues 1-263 of Figure 13, with the overlap against the specificity region in green. An 89% identity with HvYSl resulted from the protein BLAST. The Clustal2W analysis in Figure 14 shows the 5' end of the A. sativa gene as residues 1-286. There is also conservation in this area between the putative iron phytosiderophore transporter and Hv YS 1 and Zm YS 1. 26 '\IGGACGTCCTGGGCCCTGACCGCACGCGGATCGCGCCGGAGATCGAGAAGCACGTGGCCGCGGAGG GCGACAGGGAGTCTGACCCGGCGCTGGCCGCGGAGCGGGAGCTAGAGCCCCTGGGGCGGTGGCAGG ACGAGCTGACCGTGCGGGGCATGGTGGCGGCGCTGCTCATCGGGTTCATCTACACCGTCATCGTCATG AAGATCGCGCTCACCACCGGGCTGGTGCCCACCCTCAACGTCTCCGCCGCGCTGCTCTCCTTCCTCGC GCTCCGCGGCTGGACGCGCTTGCTGGACCGCTTCGGCATCGTGTCCCGTCCCTTCACGCGGCAGGAGA ACACCATCGTCCAGACCTGCGGCGTCGCCTGCTACACCATCGCGTTCGCCGGTGGCTTCGGGTCAACC TTGCTGGGTCTAAACAAGAACACGTACGAGCTGGCCGGCGACTCGCCGGGCAACGGGCCGGGGAGCT ACAAGGAGCCAGGGATTGGCTGGATGACGGCATTCCTCTTTTCTTGCAGCTTCGGGGGGCTCCTCACC TTGATTCCCCTTAGACAGGTATTGGTCGTGGACTATAAATTAGTGTACCCAAGTGGGACGGCAACTGC TGTTCTTATA,~~CGGATTTCATACCGCTCAAGGAGACAAGAACTCCAGGAAGCAAATCCGTGGGTTCT TGAAGTACTTCGGGGGTAGCTTTTTATGGAGCTTCTTCCAGTGGTTCTACACCGGCGGCGACGTTTGT GGGTTCATTCAGTTCCCTACTTTTGGTCTCAAGGCCTGGAAGCAGAC ,\ (_ ( \ ( ( ' ( ( \ \ \ \ I ( I ! ! ( (_ ( \ ( \ \ ( I { l \ \ \ I I '( ' I \ ( \( ('I \('I f \ I \( GCTATAACTATGG CAAGATAGGGCTCTTCGTCTTCGCGGGTTGGGCTGGCAGGGACAATGGTGTCGTTGCAGGTCTGGTTG TTGGTACATGTGTGAAGCAGCTGGTGCTGATATCTGCAGATTTGATGCAAGACTTCAAGACGAGTTAT CTCACTAAGACATCACCAAGATCCATGATGGTGGCACAGGCAATTGGGACAGCCATGGGCTGCGTTG TCTCTCCCCTTACGTTCATGCTCTTCTACAGGGCATTTGATATTGGCAATCCAGATGGTACCTGGAAGG CACCGTATGCACTGATATACCGTAATATGGCAATACTCGGTGTGGAGGGCTTCTCAGTACTGCCCAAG TATTGCCTGGCACTCTCTGGTGGATTTTTCGCGTTTGCAGCAATCCTCAGCATAGCAAGAGATTTCATG CCGCATAGGTATAGGCAGTATGTGCCCCTGCCAATGGCGATGGCGGTTCCATTCCTTGTCGGCGGGAG CTTTGCGATTGATATGTGTGTCGGGAGTTTGGTGGTTTTTATCTGGAACAAGATAAACAAGAAGGAGG CCGGCTTCATGGTCCCTGCAGTTGCATCCGGTTTGATATGTGGGGATGGGATATGGACATTCCCTTCG TCCATACTTGCTCTTGCCAAGATTACACCACCAATTTGCATGAAGTTTACACCTGCACCCTAG Figure 10. Full gene analysis, Nucleotide sequence. The full gene analysis of A. sativa putative ironphytosiderophore transporter. Turquoise = start codon; maroon = 5' end; green = overlap between 5' end and specificity region; pink= specificity region; gold= overlap between specificity region and 3' end; blue = 3' end of gene; red = stop codon. 27 Hordeum vulgare HvYSl mRNA for iron-phytosiderophore transporter, complete eds Length=2430 Score= 2165 bits (1172), Expect (2%) Strand=Plus/Plus Avena 1 HvYSl Avena 0.0, Identities 1763/2048 (86%), Gaps 42/2048 169 ATGGACGTCCTGGGCCCTGACCGCACGCGGATCGCGCCGGAGATCGAGAAGCACGTGGCC f I I I I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I ATGGACATCGTCGCCCCGGACCGCACGCGGATCGCGCCGGAGATCGACAGGGACGAGGCC 228 61 GC-GGAGGGCGACAGGGAGTCTGACCCGGCGCT-G-G-C--CGCG-GAGCGGGAGCTAGA 113 I 111111111111111111 11111111111 I I I 1111 Ill II 60 1111 II HvYSl 229 -CTGGAGGGCGACAGGGAGTCGGACCCGGCGCTGGCGTCGACGCGCGAGTGGCAGCTGGA 287 Avena 114 GCCCCTGGGGCGGTGGCAGGACGAGCTGACCGTGCGGGGCATGGTGGCGGCGCTGCTCAT 173 I I II 11111111111111111111 111111111 1111111111111111111 HvYSl 288 GGACATGCCACGGTGGCAGGACGAGCTGACGGTGCGGGGCCTGGTGGCGGCGCTGCTCAT 347 Avena 174 CGGGTTCATCTACACCGTCATCGTCATGAAGATCGCGCTCACCACCGGGCTGGTGCCCAC 233 1111111111111111111111111111111 f 1111111111111 11111111111111 HvYSl 348 CGGGTTCATCTACACCGTCATCGTCATGAAGATCGCGCTCACCACGGGGCTGGTGCCCAC 407 Avena 234 CCTCAACGTCTCCGCCGCGCTGCTCTCCTTCCTCGCGCTCCGCGGCTGGACGCGCTTGCT 293 II l l l l l l l l l l l l l l l l l l l l l l l l l l l l l 11111111111 111111111 1111 HvYSl 408 GCTGAACGTCTCCGCCGCGCTGCTCTCCTTCCTGGCGCTCCGCGGGTGGACGCGCCTGCT 467 Avena 294 GGACCGCTTCGGCATCGTGTCCCGTCCCTTCACGCGGCAGGAGAACACCATCGTCCAGAC 353 111 111111111 1111111111 11111111111111111111111111111111111 HvYSl 468 GGAGCGCTTCGGCGTCGTGTCCCGCCCCTTCACGCGGCAGGAGAACACCATCGTCCAGAC 527 Avena 354 CTGCGGCGTCGCCTGCTACACCATCGCGTTCGCCGGTGGCTTCGGGTCAACCTTGCTGGG 413 111111111 II 11111111111111111 11111111 11111111111111111111 HvYSl 528 CTGCGGCGTGGCATGCTACACCATCGCGTTTGCCGGTGGGTTCGGGTCAACCTTGCTGGG 587 Avena 414 TCTAAACAAGAACACGTACGAGCTGGCCGGCGACTCGCCGGGCAACGGGCCGGGGAGCTA 473 II 11111111 11111111111111111 1111111111111111 Ill II till HvYSl 588 CCTGAACAAGAAGACGTACGAGCTGGCCGGTGACTCGCCGGGCAACGTGCCCGGAAGCTG 647 Avena 474 CAAGGAGCCAGGGATTGGCTGGATGACGGCATTCCTCTTTTCTTGCAGCTTCGGGGGGCT 533 11111111111111 1111111111111 111111 I 1111111111111111 II HvYSl 648 GAAGGAGCCAGGGATAGGCTGGATGACGGGGTTCCTCCTCGCTTGCAGCTTCGGGGGCCT 707 Avena 534 CCTCACCTTGATTCCCCTTAGACAGGTATTGGTCGTGGACTATAAATTAGTGTACCCAAG 593 111111 1111111 It 11 111111111 1111111 11111 t 1111111 11111111 HvYSl 708 CCTCACTTTGATTCCCCTGAGACAGGTACTGGTCGTCGACTACAAATTAGTTTACCCAAG 767 Avena 594 TGGGACGGCAACTGCTGTTCTTATAAACGGATTTCATACCGCTCAAGGAGACAAGAACTC 653 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I I I I I I I I I II I I I I I I HvYSl 768 TGGGACTGCAACTGCTATTCTTATAAACGGGTTCCATACCGATCAAGGGGACAAGAATTC 827 Avena 654 CAGGAAGCAAATCCGTGGGTTCTTGAAGTACTTCGGGGGTAGCTTTTTATGGAGCTTCTT 713 II HvYSl 828 11111111111111 Ill 1111 11111 111111111111 I 11111 11111 AAGAAAGCAAATCCGTGGATTCCTGAAATACTTTGGGGGTAGCTTTCTGTGGAGTTTCTT 887 28 Avena 714 HvYSl 888 Avena 774 HvYSl 948 Avena 834 HvYSl 1008 Avena 892 HvYSl 1065 Avena 950 HvYSl 1124 Avena 1010 HvYSl 1184 Avena 1068 HvYSl 1242 Avena 1127 HvYSl 1301 Avena 1186 HvYSl 1360 Avena 1244 HvYSl 1418 Avena 1304 HvYSl 1478 Avena 1364 HvYSl 1538 Avena 1424 HvYSl 1598 Avena 1483 HvYSl 1657 CCAGTGGTTCTACACCGGCGGCGACGTTTGTGGGTTCATTCAGTTCCCTACTTTTGGTCT 111 11111111111 11 11111 I 111111 11 1111111111 11111 111 I CCAATGGTTCTACACTGGAGGCGATGCTTGTGGATTTGTTCAGTTCCCAACTTTCGGTTT 773 CAAGGCCTGGAAGCAGACGTTCTTCTTTGACTTTAGCCTGACATACATCGGTGCCGGGAT I I I I I I I l I I l I I I I I I 111 I 1111111111111 11111111 I 11111111111 GAAGGCCTGGAAGCAGACATTCTACTTTGACTTTAGCATGACATACGTTGGTGCCGGGAT 833 GATCTGCCCACATATAGTAAATATCTCCACCCTC-TTGGGTGCAATTCTTTCTTATGGGI I I I I I 11 I I I I I I I I 11 I I I I I I I I I I I I I I I I II I I I I I I I I I I I I I GATTTGTCCACATATAGTAAATATATCTACCCTCCTTGG-TGCAATTATCTC--ATGGGG 891 A-TATTGTGGCCACTCATCAGTAAGAACAAGGGTGACTGGTACCCTGCAGATGT-CAAAG I I I I I I II I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I AATAATGTGGCCACTCATCAGTAAAAACAAGGGGGATTGGTACCCTGCAAAAGTACCA-G 949 947 1007 1064 1123 AAAGCAGCATGAAAAGTTTGTACGGTTACAAGGCCTTCATATGCATCGCTCTGATCATGG 1111111111111111111111111111111111111111111111 11111 1111111 AAAGCAGCATGAAAAGTTTGTACGGTTACAAGGCCTTCATATGCATAGCTCTCATCATGG 1009 GGGATGG-ACTCTACCACTTCACCAAAATTATTACCATCACTTGCAAGG-GCATGTATCG I I I I I I I I I 111 I I I I I I I IIIIII II I I I I I I I I I I I I I I I I I II I I GGGATGGCA-TGTACCACTTCATAAAAATTGTTGGCATCACT-GCTATGAGCATGTATCG 1067 ACAGTTCAGCCGTAAACATGCTGACAACCGAG-AGAAAAATGTGGACAATACAGTCTCAC II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I GCAATTTAGCCACAAGCAGGTTAACAACAAAGCA-AAAAATGCGGACGACACTGTCTCGC 1126 1183 1241 1300 TCGAGGATTTGCAGCGCGACGAGGTCTTCAAGAGGGGCCATC-TCCCCGCTTGGATCGCG I 11111 II II Ill I Ill 1111 11111 11111 I 11111 1111111 II TTGAGGAGTTACACCGCCAGGAGATCTTTAAGAGAGGCCA-CATCCCCTCTTGGATGGCA 1185 TACAG-TGGGTATGCCGTGCTGAGCGT-CGTTGCAGTGGTTACCACGCCAATAATGTTCC Ill I Ill 111111 II I 11111 I l l l l l l l l l l l l l I Ill 11111111 TAC-GCTGGTTATGCCTTGTTTAGCGTTC-TTGCAGTGGTTACAATACCAGTAATGTTCA 1243 GACAAGTGAAATGGTACTACGTAGTTATAGCCTATGTCGTCGCCCCCATGCTTGGATTCG 111111111 11111111 II l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l I AACAAGTGAAGTGGTACTATGTTGTTATAGCCTATGTCGTTGCCCCCATGCTTGGATTTG 1303 1359 1417 1477 CCAATTCCTACGGGACGGGGCTCACCGACATCAACATGGGCTATAACTATGGCAAGATAG 1111111 11111111 11111 l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l I CCAATTCATACGGGACAGGGCTTACCGACATCAACATGGGCTATAACTATGGCAAGATCG 1363 GGCTCTTCGTCTTCGCGGGTTGGGCTGGCAGGGACAATGGTGTCGTTGCAGGTCTGGTTG 11111 11111 11111 11111 11 I II l l l l l l l l l 1111 II II 1111 CTCTCTTTGTCTTTGCGGGATGGGCCGGTAAAGAGAATGGTGTCATTGCCGGCCTTGTTG 1423 TTGGTACATGTG-TGAAGCAGCTGGTGCTGATATCTGCAGATTTGATGCAAGACTTCAAG I I I I I I I I I I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I I II I I I I I I CTGGCACCT-TGGTTAAGCAGTTGGTGCTTATCTCTGCCGATTTGATGCAAGACTTCAAG 1482 ACGAGTTATCTCACT-AAGACATCACCAAGATCCATGATGGTGGCACAGGCAATTGGGAC 11111111 111111 II 1111111111 1111111111 I 11111 I 1111 ti ACGAGTTACCTCACTCAA-ACATCACCAAAATCCATGATGATTGCACAAGTTGTTGGAAC 1537 1597 1656 1541 1715 29 Avena 1542 HvYSl 1716 Avena 1602 HvYSl 1776 Avena 1662 HvYSl 1836 Avena 1720 HvYSl 1894 Avena 1779 HvYSl 1953 Avena 1839 HvYSl 2013 Avena 1898 HvYSl 2072 Avena 1958 HvYSl 2132 Avena 2018 AGCCATGGGCTGCGTTGTCTCTCCCCTTACGTTCATGCTCTTCTACAGGGCATTTGATAT 111111111 111 1111111 11111 1111111111111111111 111111111111 AGCCATGGGTTGCATTGTCTCCCCCCTCACGTTCATGCTCTTCTACAAGGCATTTGATAT 1601 TGGCAATCCAGATGGTACCTGGAAGGCACCGTATGCACTGATATACCGTAATATGGCAAT 111 11 11111111111 11111111111 11111111 11 I 11111 I I 111111111 TGGTAACCCAGATGGTACTTGGAAGGCACCTTATGCACTCATATACCGCAATATGGCAAT 1661 ACTCGGTGTGGAGGGCTTCTCAGTACTGCCCAAGTATTGCCT-G-GCACTCTCTGGTGGA 111 11111111111111111 11 1111 111111111 I I I I I 11 111111 ACTTGGTGTGGAGGGCTTCTCGGTGTTGCCGAAGTATTGCATCGTG-A-TATCCGGTGGA 1719 TTTTTCGCGTTTGCAGCAATCCTCAGCATAGCAAGAGATTTCATGCCGCATAGGTATAG11111111 I 1111 11 11 I 11 111 11111111 1111111 11 I 1111 I TTTTTCGCCTTTGCGGCGATTTTAAGTATAACAAGAGATGTCATGCCCCACAAGTAT-GC 1778 GCAGTATGTGCCCCTGCCAATGGCGATGGCGGTTCCATTCCTTGTCGGCGGGAGCTTTGC I IIIIIIIIIIIIIIIIIIIIII 11111 11111111111111 II 11111111111 GAAGTATGTGCCCCTGCCAATGGCAATGGCAGTTCCATTCCTTGTAGGTGGGAGCTTTGC 1838 GATTGATATGTGTGTCGGGAGTTTGGTGGTTTTTATC-TGGAACAAGATAAACAAGAAGG 11111111111 11111111111 I 111111 I 1111 11111111111111111 TATTGATATGTGCCTCGGGAGTTTGATAGTTTTTG-CATGGACCAAGATAAACAAGAAGG 1897 AGGCCGGCTTCATGGTCCCTGCAGTTGCATCCGGTTTGATATGTGGGGATGGGATATGGA I I I I I I I I I 11 111 I I I I I I I I I I 11 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I AGGCTGGCTTCATGGTGCCTGCGGTTGCATCCGCTTTGATATGTGGGGATGGCATATGGA 1957 CATTCCCTTCGTCCATACTTGCTCTTGCCAAGATTACACCACCAATTTGCATGAAGTTTA I 111111 I 11111 1111 I 11111111111111 1111111111111111111111 CGTTCCCTGCTTCCATTCTTGCTCTTGCCAAGATTAAACCACCAATTTGCATGAAGTTT- 2017 1775 1835 1893 1952 2012 2071 2131 2190 C-ACCTGC 2024 I 111111 HvYSl 2191 CTACCTGC 2198 Figure 11: Full gene nucleotide BLAST of Avena iron phytosideropbore putative transporter. Full nucleotide BLAST of A. sativa putative iron phytosiderophore transporter. 1-800 represents the 5' end, 793-1324 represents the specificity region, 1231-2024 represents the 3' end of the gene. 'vlDVLGPDRTRIAPf:.IEKHVAALGDRcSDPALAAI,RI'.Lf-:Pl GRWQDf,LTVRGMVAAl l lGFiYTVI VMK!Al r Hr! VPTI '\JV'-AALl ~FLAl RGWTRl I DRFGIYSRPF l'ROFNTIYOTCGVACY r (AJ'.. AGGF'(;~l Ll (d NK '1 r 1 ELAGDSPG~GPGSYKEPGIGWYI I AH FSCSFGGL l 1 Li Pl RQVI YVDYKL YYPSG l'A I A YLlNGFHTAQCi Dh.'\i~RKOIRGFl KYFGGSrtw-::HQWFYTGGDVCGFIOF'PTFCil KA WK Q"I FF I >I \I 'ill \(,\11( l'I 1 1\ \ I 'y(, \ )'\RI ! ','-!· ;,~',1'<, )',\'if>\ )\i,. ,,'\H,'-.i ,,.,,, \ill \l''\h."il I \11 1 l~-1 "If( [ 11\I\K_(l1 r-. '\\ ) LTDINMGYNYGK.IGLFVF AGWAGRDNGVV AGL VVGTCVKQLVLISADLMQDFK TSYLTKTSPRSMMVAQAIGTAMGCVVSPLTFMLFYRAFDIGNPDGTWKAPY ALIYRNMAILGVEGFSVLP KYCLALSGGFF AF AAILSIARDFMPHRYRQYVPLPMAMAVPFLVGGSF AIDMCVGSLVVFIWNK.INKKEA GFMVPAVASGLICGDGIWTFPSSILALAK.ITPPICMKFTPAP Figure 12: Full gene protein analysis of putative Avena iron phytosiderophore transporter. 30 iron-phytosiderophore transporter [Hordeum vulgare subsp. vulgare] Length=678 Score= 1230 bits (3182), Expect= 0.0, Method: Compositional matrix adjust. Identities= 583/677 (86%), Positives= 633/677 (93%), Gaps= 2/677 (0%) Avena 1 HvYSl 1 Avena 59 HvYSl 61 Avena 119 HvYSl 121 Avena 179 HvYSl 181 Avena 239 HvYSl 241 Avena 299 HvYSl 301 Avena 359 HvYsl 361 Avena 419 HvYSl 421 Avena 479 HvYSl 481 Avena 539 HvYSl 541 Avena 599 HvYSl 601 Avena 659 HvYSl 661 MDVLGPDRTRIAPEIEKHVAAEGDRESDPALAAERE--LEPLGRWQDELTVRGMVAALLI MD++ PDRTRIAPEI++ A EGDRESDPALA+ RE LE+ RWQDELTVRG+VAALLI MDIVAPDRTRIAPEIDRDEALEGDRESDPALASTREWQLEDMPRWQDELTVRGLVAALLI 58 GFIYTVIVMKIALTTGLVPTLNVSAALLSFLALRGWTRLLDRFGIVSRPFTRQENTIVQT GFIYTVIVMKIALTTGLVPTLNVSAALLSFLALRGWTRLL+RFG+VSRPFTRQENTIVQT GFIYTVIVMKIALTTGLVPTLNVSAALLSFLALRGWTRLLERFGVVSRPFTRQENTIVQT 118 CGVACYTIAFAGGFGSTLLGLNKNTYELAGDSPGNGPGSYKEPGIGWMTAFLFSCSFGGL CGVACYTIAFAGGFGSTLLGLNK TYELAGDSPGN PGS+KEPGIGWMT FL +CSFGGL CGVACYTIAFAGGFGSTLLGLNKKTYELAGDSPGNVPGSWKEPGIGWMTGFLLACSFGGL 178 LTLIPLRQVLVVDYKLVYPSGTATAVLINGFHTAQGDKNSRKQIRGFLKYFGGSFLWSFF LTLIPLRQVLVVDYKLVYPSGTATA+LINGFHT QGDKNSRKQIRGFLKYFGGSFLWSFF LTLIPLRQVLVVDYKLVYPSGTATAILINGFHTDQGDKNSRKQIRGFLKYFGGSFLWSFF 238 QWFYTGGDVCGFIQFPTFGLKAWKQTFFFDFSLTYIGAGMICPHIVNISTLLGAILSYGI QWFYTGGD CGF+QFPTFGLKAWKQTF+FDFS+TY+GAGMICPHIVNISTLLGAI+S+GI QWFYTGGDACGFVQFPTFGLKAWKQTFYFDFSMTYVGAGMICPHIVNISTLLGAIISWGI 298 LWPLISKNKGDWYPADVKESSMKSLYGYKAFICIALIMGDGLYHFTKIITITCKGMYRQF +WPLISKNKGDWYPA V ESSMKSLYGYKAFICIALIMGDG+YH MWPLISKNKGDWYPAKVPESSMKSLYGYKAFICIALIMGDGMYHFIKIVGITAMSMYRQF 358 60 120 180 240 300 360 SRKHADNREKNVDNTVSLEDLQRDEVFKRGHLPAWIAYSGYAVLSVVAVVTTPIMFRQVK 418 AVVT P+MF+QVK ' SHKQVNNKAKNADDTVSLEELHRQEIFKRGHIPSWMAYAGYALFSVLAVVTIPVMFKQVK 420 WYYVVIAYVVAPMLGFANSYGTGLTDINMGYNYGKIGLFVFAGWAGRDNGVVAGLVVGTC WYYVVIAYVVAPMLGFANSYGTGLTDINMGYNYGKI LFVFAGWAG++NGV+AGLV GT WYYVVIAYVVAPMLGFANSYGTGLTDINMGYNYGKIALFVFAGWAGKENGVIAGLVAGTL 478 VKQLVLISADLMQDFKTSYLTKTSPRSMMVAQAIGTAMGCVVSPLTFMLFYRAFDIGNPD VKQLVLISADLMQDFKTSYLT+TSP+SMM+AQ +GTAMGC+VSPLTFMLFY+AFDIGNPD VKQLVLISADLMQDFKTSYLTQTSPKSMMIAQVVGTAMGCIVSPLTFMLFYKAFDIGNPD 538 GTWKAPYALIYRNMAILGVEGFSVLPKYCLALSGGFFAFAAILSIARDFMPHRYRQYVPL GTWKAPYALIYRNMAILGVEGFSVLPKYC+ +SGGFFAFAAILSI RD MPH+Y +YVPL GTWKAPYALIYRNMAILGVEGFSVLPKYCIVISGGFFAFAAILSITRDVMPHKYAKYVPL 598 PMAMAVPFLVGGSFAIDMCVGSLVVFIWNKINKKEAGFMVPAVASGLICGDGIWTFPSSI PMAMAVPFLVGGSFAIDMC+GSL+VF W KINKKEAGFMVPAVAS LICGDGIWTFP+SI PMAMAVPFLVGGSFAIDMCLGSLIVFAWTKINKKEAGFMVPAVASALICGDGIWTFPASI 658 LALAKITPPICMKFTPA LALAKI PPICMKF PA LALAKIKPPICMKFLPA 480 540 600 660 675 677 Figure 13: Full Gene Protein BLAST of putative Avena transporter. Protein BLAST of A. sativa putative iron- phytosiderophore transporter against best match HvYSl. Specificity area shown in pink. 31 Avena HvYSl ZmYSl MDVLGPDRTRIAP---EIEKHVAAEGDRESDPALAAER--ELEPLGRWQDELTVRGMVAA 55 MDIVAPDRTRIAP---EIDRDEALEGDRESDPALASTREWQLEDMPRWQDELTVRGLVAA 57 MDLARRGGAAGADDEGEIERHEPAPEDMESDPAAAREKELELERVQSWREQVTLRGWAA 60 * ***** * **: * **: ·** *:: :*:**:*** Avena HvYSl ZmYSl LLIGFIYTVIVMKIALTTGLVPTLNVSAALLSFLALRGWTRLLDRFGIVSRPFTRQENTI 115 LLIGFIYTVIVMKIALTTGLVPTLNVSAALLSFLALRGWTRLLERFGWSRPFTRQENTI 117 LLIGFMYSVIVMKIALTTGLVPTLNVSAALMAFLALRGWTRVLERLGVAHRPFTRQENCV 120 *****:*:**********************::*********:*:*:*:. ******** Avena HvYSl ZmYSl VQTCGVACYTIAFAGGFGSTLLGLNKNTYELAGDSPGNGPGSYKEPGIGWMTAFLFSCSF 175 VQTCGVACYTIAFAGGFGSTLLGLNKKTYELAGDSPGNVPGSWKEPGIGWMTGFLLACSF 177 IETCAVACYTIAFGGGFGSTLLGLDKKTYELAGASPANVPGSYKDPGFGWMAGFVAAISF 180 ::**.********.**********:*:****** **.* ***:*:**:***:.*: : ** Avena HvYSl ZmYSl GGLLTLIPLRQVLWDYKLVYPSGTATAVLINGFHTAQGDKNSRKQIRGFLKYFGGSFLW 235 GGLLTLIPLRQVLWDYKLVYPSGTATAILINGFHTDQGDKNSRKQIRGFLKYFGGSFLW 237 AGLLSLIPLRKVLVIDYKLTYPSGTATAVLINGFHTKQGDKNARMQVRGFLKYFGLSFVW 240 ***:*****:***:****.********:******* *****:* *:******** **:* Avena HvYSl ZmYSl SFFQWFYTGGDVCGFIQFPTFGLKAWKQTFFFDFSLTYIGAGMICPHIVNISTLLGAILS 295 SFFQWFYTGGDACGFVQFPTFGLKAWKQTFYFDFSMTYVGAGMICPHIVNISTLLGAIIS 297 SFFQWFYTGGEVCGFVQFPTFGLKAWKQTFFFDFSLTYVGAGMICSHLVNISTLLGAILS 300 **********:.***:**************:****:**:******.*:**********:* Avena HvYSl ZmYSl YGILWPLISKNKGDWYPADVKESSMKSLYGYKAFICIALIMGDGLY WGIMWPLISKNKGDWYPAKVPESSMKSLYGYKAFICIALIMGDGMY ' WGILWPLISKQKGEWYPANIPESSMKSLYGYKAFLCIALIMGDGTY _ _ __ :**:******:**:****.: *************:********* *** *· ·* r Avena HvYSl ZmYSl ,I , r•· ;.,: ::*:*: *: * *: :*::*:*:*:* * :*:* **:*** :*: * ) '•,•:1 ' ,; ' I~ 355 357 360 PIMF 414 PVMF 416 PHMF 420 * ** Avena HvYSl ZmYSl RQVKWYYWIAYWAPMLGFANSYGTGLTDINMGYNYGKIGLFVFAGWAGRDNGWAGLV 474 KQVKWYYWIAYWAPMLGFANSYGTGLTDINMGYNYGKIALFVFAGWAGKENGVIAGLV 476 RQVKWYYVIVAYVLAPLLGFANSYGTGLTDINMAYNYGKIALFIFAAWAGRDNGVIAGLA 480 :*******::***:**:****************.******.**:**.***::***:*** Avena HvYSl ZmYSl VGTCVKQLVLISADLMQDFKTSYLTKTSPRSMMVAQAIGTAMGCWSPLTFMLFYRAFDI 534 AGTLVKQLVLISADLMQDFKTSYLTQTSPKSMMIAQWGTAMGCIVSPLTFMLFYKAFDI 536 GGTLVKQLVMASADLMHDFKTGHLTMTSPRSLLVAQFIGTAMGCWAPLTFLLFYNAFDI 540 ** *****: *****:****.:** ***:*:::** :******:*:****:***.**** Avena HvYSl ZmYSl GNPDGTWKAPYALIYRNMAILGVEGFSVLPKYCLALSGGFFAFAAILSIARDFMPHRYRQ 594 GNPDGTWKAPYALIYRNMAILGVEGFSVLPKYCIVISGGFFAFAAILSITRDVMPHKYAK 596 GNPTGYWKAPYGLIYRNMAILGVEGFSVLPRHCLALSAGFFAFAFVFSVARDVLPRKYAR 600 *** * *****.******************::*:.:*.****** ::*::**.:*::* Avena HvYSl ZmYSl YVPLPMAMAVPFLVGGSFAIDMCVGSLWFIWNKINKKEAGFMVPAVASGLICGDGIWTF 654 YVPLPMAMAVPFLVGGSFAIDMCLGSLIVFAWTKINKKEAGFMVPAVASALICGDGIWTF 656 FVPLPMAMAVPFLVGGSFAIDMCVGSLAVFVWEKVNRKEAVFMVPAVASGLICGDGIWTF 660 :**********************:*** ** * *:*:*** ********.********** 32 Avena HvYSl ZmYSl PSSILALAKITPPICMKFTPAP 676 PASILALAKIKPPICMKFLPAA 678 PSSILALAKIKPPICMKFTPGS 682 *:********.******* * Figure 14. Clustal analysis between known transporter and putative Avena tranporter. Full gene Clustal2W analysis of Avena putative iron-phytosiderophore transporter against published transporters HvYSl and ZmYSl . 33 Discussion: The isolated gene from A. sativa is 2,031 nucleotides in length and the protein is 676 amino acids in length. Based on nucleotide BLAST results, the iron-phytosiderophore transporter from A. sativa showed 86% and 77% identity to HvYSl and Zm YSl, respectively. A protein BLAST of the sequence from Avena sativa and the two confirmed transporters showed an OPT domain, providing more evidence of similarity. The closer similarity to Hv YS 1 is also suggestive of the iron specificity of the transporters and the broad specificity of Zm YSl, which has been tested in various experiments (Meda et al. 2007; Roberts et al. 2004; Chauhan 2006; Yen et al. 2001; Schaaf et al. 2004; von Wiren et al. 1996). This diversity can be seen in the nucleotide and amino acid sequences in the area called the "specificity region"; further verification of this diversity can be seen when comparing the sequences to A. stolonifera, which exhibits non-similarity in the same region. The difference in the substrate specificity between HvYSl and Zm YSl may result from the stereostructure of the transport protein. HvYSl has 11 putative transmembrane spanning domains while Zm YS 1 has 12 putative transmembrane spanning domains (Murata et al. 2006). The discovery of the iron-phytosiderophore transporter in a major grain crop, oats, opens the door for further analysis and engineering into enhancement of iron uptake by non-graminaceous transformants. Increasing the iron concentration of plant foods will require an increase in total iron input to the plant that can potentially be achieved through genetic manipulation of the plant itself. Different processes that can be targeted for genetic transformation include root iron acquisition, transport through the vascular tissues, and storage in edible tissues (Vasconcelos and Grusak 2006). Understanding this iron phytosiderophore transporter system may allow the development of a system to deliver effector molecules with specificity to Avena or a transformed target species using phytosiderophore conjugates and the transporter as a portal through which to deliver the effector. This approach has not been investigated elsewhere, but is currently underway in the Davis laboratory. Other areas that are being considered are the biofortification of major seed and crop plants, phytoremediation strategies, and re-greening of non-graminaceous plants. 34 WHO recommends enriching plants with iron - a concept termed "Iron Biofortification." Plant grains are one of the major sources for iron in the human diet, especially in poor countries where anemia is prevailing. However, the current mechanisms controlling iron homeostasis in plants are not completely understood. Once these processes are clearer, there will be a starting point for better manipulation of crop species (Briat 2008; Gross et al. 2003; World Health Organization; Xiong and Ishitani 2006). Biofortification is a process whereby the plant uses its own mechanisms to fortify or enhance the density or bioavailability of nutrients (like iron) in its edible tissues. The development of iron biofortified plants involves four main strategies: (1) cultivar evaluation, (2) plant breeding and marker assisted selection, (3) alteration of pathways of iron metabolism, and (4) modification of iron bioavailability (Vasconcelos and Grusak 2006). This process is not a new idea because fortified products such as infant formula and iron enriched flours are already available. However, by fortifying certain plant species before harvest, there is I a higher probability of getting iron fortified products to where they are needed most (Guerinot 2001 ). There is the possibility that phytosiderophores released by graminaceous species can mobilize iron from sparingly soluble soil sources; the Fe3+ thus released can be taken up by dicots after reduction. Results from studies might explain the beneficial effects of intercropping dicots with grasses in order to supply Strategy I plants with iron from the rhizosphere. Cesco et al showed that the amount of 59Fe taken up by iron deficient citrus plants increased when iron deficient barley plants were present in the uptake solution, particularly in the case of the susceptible rootstock. Enhanced 59Fe uptake by citrus rootstocks was also observed in a separate experiment, where the authors collected exudates from the iron deficient barley plants during the period of high phytosiderophore release (morning) were added to the solution (Cesco et al. 2006). Re-greening of fruit trees observed in calcareous soils when grass is grown along the rows of trees has been attributed to the capability of graminaceous plant species to increase the availability of iron by releasing phytosiderophores into the soil, thus favoring the uptake ofthis micronutrient by the fruit trees. The results indicated that fruit trees growing on calcareous soils can benefit, in terms of iron nutrition, from intercropping with perennial grasses that increase iron availability for root uptake. 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