EXPRESSION OF AtZIP1 INCREASE OF ZINC CONCENTRATIONS IN TOBACCO KAHYA SHUAIBU 1,2,3, Narayanan Narayanan 1, Eliana Gaitan-Solis 1, Chiedozie Egesi ², Joseph Onyeka ², Emmanuel Okogbenin ², E.N.A. Mbanaso², F.I.Onyenobi ³, Martin Fregene¹ and Richard Sayre 1 1 Donald Danforth Plant Science Center, St. Louis, MO 63132, USA ² National Root Crops Research Institute Umudike, P.M.B 7006,Umuahia, Abia state,Nigeria ³ Abia state university Uturu,Facaulty of Agriculture and Veterinary Medicine Umudike Umuahia Campus ABSTRACT METHODS SCHEMATIC PROCEDURE FOR TOBACCO REGENERATION PATH OF TRANSITION METALS AND GENETIC ENGINEERING TARGET INTRODUCTION 1. CONCLUSIONS Use Uptake Phloem transport Storage and detoxification (d) Storage and detoxification (a) Mobilization (b) Uptake (c) Xylem loading Symplastic passage Symplastic passage Xylem transport Apoplastic passage unloading PCR AMPLIFICATION CLONING DIGESTION SEQUENCING AGRO-TRANSFORMATION TOBACCO LEAF DISKS Transition metal from the soil to the sites of use and storage in the leaf. (a) to enhance mobilization by secretion of organic acids, (b) to increase uptake by over expression or deregulation of transporters, (c) to stimulate uptake into the root and translocation via the xylem by overproduction of intracellular chelators, (d) to increase the strength of metal sinks in the leaves by over expression of storage and detoxification mechanisms. OBJECTIVE (Stephan et,al;2002) A14-AtZIP1-tNOS construct in p2301 was given as a gift from Eliana Gaitan-solis, DDPSC. Primers with restriction enzymes (EcoRI and KpnI) were designed to pull out the construct and cloned it in pCAMBIA2300. AtZIP1 driving by A14- root epidermal promoter was introduced into cassava (FEC) via Agrobacterium - mediated transformation. A14-AtZIP1- tNOS construct was introduced into tobacco through leaf disk via Agrobacterium –mediated transformation. Transgenic tobacco lines were screened by PCR, RT-PCR and dot blot analysis to confirm the presence of the gene. Inductively Couple Plasma Mass Spectrophotometry(ICP-MS) were carried out for transgenic and wild type seeds, leaves and roots. The seeds were sterilized and germinated on 150 mg/l kanamycin plates (MS0- media) for each generation. S breeding for higher Zn in cassava may not be feasible. Current knowledge on the molecular and biochemical mechanisms of transition metal homeostasis is commonly used to modify metal uptake, root -to- shoot translocation, and distribution at the cellular, tissue, and organ levels. Such alterations are focus to enhance crops for higher mineral levels. Biofortification aims at an efficient micronutrient uptake mainly from poor soils, and an efficient translocation to the edible parts of crop plants (Kramer et al.,2007; Palmgren et al., 2008; Kramer, 2009). through genetic engineering as a promising tool. Zinc(Zn) is essential in plants, animals, and humans. However, it is frequently deficient in the diet, resulting in poor health. Across the world, there are many soils that are Zn-deficient or with low Zn bioavailability. Consequently, crops cultivated there contain low Zn concentrations leading to Zn- deficiency-based malnutrition. The mechanisms to enhance Zn accumulation and translocation of Zn in crop plants is still rudimentary. Here in this study, we used tobacco (Nicotiana tabacum) as a model crop to study the expression of a zinc transporter from Arabidopsis (AtZIP1). Tobacco has several advantages, including a high biomass, moderate soil requirements, fast growth rate, ease of harvesting and not a metal hyperaccumulator. ICP-MS ANALYSIS A Rooting media Selection T ₀ Generation Soil Germination media T1 Generation Seeds Mn Conc. (mg/kg) Fe Conc.(mg/kg)Zn Conc. (mg/kg) Cu Conc.mg/kg We would like to thank Kevin Lutke, Tissue Culture Facility, DDPSC for transforming into Tobacco. Funding from Gates Foundation and support from Biocassava plus and NRCRI Umudike is greatly appreciated  Tobacco transgenic lines carrying A14-AtZIP1-tNOS shows a promising phenotype in shoots indicating a balanced Zn homeostasis. This suggested AtZIP1 with A14 promoter may be a good strategy to maintain Zn homoestasis. These mechanism of Zn distribution and accumulation in plants will contribute to biofortification of staple food crops.  Combining this strategy with a storage gene (AtMTP1) and tuber specific promoter (Patatin) is under way. PAT-AtMTP1 is already known to accumulate zinc in the root tubers. Therefore, probably combining these two construct will balance zinc homeostasis in the plant and maintain high zinc concentration in the target root tissue.  Cassava (Manihot esculenta Crantz) is an important staple crop, especially for resource poor populations in sub-Saharan Africa which lacks micronutrients especially zinc and iron. Mimicking our strategies in cassava would enhance zinc homeostasis in the edible part of the crop and will play a major role in reducing the micronutrient malnutriton in Africa. Preliminary analysis shows that A14 is expressed in root epidermis and leaves (Elisa LevyaGuerrero, unpublished data). This should increase the transport of zinc into the root epidermis and not concentrate in the cortex there by preventing the accumulation of zinc into the root alone. ATZIP1NOS A14 STRATEGY The ICP-MS analysis shows that there is a 1.5 fold increase of Zn in the transgenic roots (Line T3 and T4) when compared with the WT. Also, few transgenic lines show a reduction of zinc in leaves compared to the wild type. Interestingly, there was no significant difference in zinc concentrations in the seeds of transgenic and WT plants. ZIP1 being a transporter of iron, we see a 3 fold increase in the transgenic root when compared with the WT. There is no significant difference between Mn and Cu distribution in the leaves, seeds, root of transgenic lines. Gene name Expression A14Epidermis, enriched in atrichoblast To understand the molecular mechanisms of zinc accumulation, distribution, sequestration in subcellular compartments of different tissues by expression of AtZIP1 using a A14 epidermal promoter. T2 Generation T3 Generation (Homozygous) ACKNOWLEDGEMENTS T1 T2 T3 T4 T5 T6 T7 T8 A14:ZIP H₂0H₂0 WT PCR OF TRANSGENIC LINES