rea: Biofortificao

Genetic components useful for iron and zinc biofortification in Vigna unguiculata (L.) Walp.
Lus Carlos Belarmino1,2,3, Ana Carolina Wanderley-Nogueira1, Lidiane Lindinalva Barbosa Amorim1, Peter Winter2, Gunter Kahl2,3 e Ana Maria Benko-Iseppon1
1

Department of Genetics, Center of Biological Sciences, Universidade Federal de Pernambuco Recife, PE, Brazil.
2 3

GenXPro GmbH, Frankfurt am Main, Germany.

Johann Wolfgang Goethe University, Frankfurt am Main, Germany. email: lucabellarmino@gmail.com

Resumo A deficincia nutricional de micronutrientes como ferro e zinco compreende um srio problema mundial, causando 0,8 milhes de mortes anualmente. Como estratgia para mudar esse cenrio, tem se proposto a biofortificao de culturas para consumo alimentar, especialmente aquelas que chegam populao pobre por um baixo custo aos cofres pblicos, fator importante sustentabilidade de programas de combate desnutrio. Devido importncia do feijo-caupi no combate desnutrio e pobreza, uma srie de estudos moleculares centrados no conjunto de genes dessa leguminosa vm sendo conduzidos no laboratrio de gentica e biotecnologia vegetal da UFPE, visando ao melhoramento de caractersticas agronmicas e nutricionais. O objetivo do presente trabalho foi investigar componentes genticos em rotas de captao, armazenamento e metabolismo de ferro e zinco nas razes de feijo-caupi. Como resultados, apresentamos a reconstruo de uma rota metablica in silico, cuja modificao tem potencial para alterar a deposio destes micronutrientes. Diante dos dados apresentados, consideramos a realizao de estudos de expresso para confirmar a co-expresso tecidual dos genes relatados e sua possvel correlao com fentipos contrastantes para o acmulo de ferro e zinco e possvel uso biotecnolgico de seus componentes gnicos.

Abstract Deficiency in iron and zinc micronutrients comprises a serious problem worldwide, causing 0,8 million deaths annually. As strategy to change this scenario, crop biofortification has been proposed as a cost-effective and sustainable approach for the governments to alleviate malnutrition. Due to the importance of cowpea in fighting malnutrition and poverty, a number of molecular studies focused on the cowpea gene set has been conducted in the Plant Genetics and Biotechnolgy Laboratory at UFPE, aiming at the improvement of agronomic and nutritional traits. The goal of the present work was to search for genetic components in iron and zinc uptake, storage and metabolism pathway in cowpea roots. As outcome, we present the in silico reconstruction of a metabolic pathway, whose modification has the potential to change the deposition of these micronutrients. From the exposed data, we consider future expression assays to confirm the tissue co-expression of the reported genes and their correlation with the contrasting iron and zinc accumulation phenotypes and their possible biotechnological use of their genic components. Key-words: Iron/Zinc fertilizers, Biofortification, Micronutrient bioavailability Introduction Plant biofortification is being increasingly recognized as an efficient and cost-effective strategy for reducing micronutrient malnutrition in the developing world. Assays with biofortified staples in terms of averted disabilityadjusted life years (DALY) showed that the reduction in burden of vitamin A, iron and zinc can reach up to 32%, 36% and 56%, respectively (Meenakshi et al., 2010). In humans, vitamin A is essential for vision, immune response, epithelial cell growth, bone growth, reproduction, embryonic development, and gene regulation. Likewise, iron is essential for preventing anemia and for proper functioning of many metabolic processes while zinc is essential for

adequate growth, sexual maturation, and resistance to gastro-enteric and respiratory infections, notably in children (Bouis, 2003). Cowpea [Vigna unguiculata (L.) Walp.] is an edible legume with relatively high iron and zinc content. A number of studies have demonstrated a considerable genetic variability underlying different accumulation levels of these important micronutrients, pointing some parental lines to be considered for breeding programs aiming at iron and zinc biofortification (Carvalho et al., 2011; Mamiro et al., 2011; Moura et al., 2011, 2012). These resources have the potential to enable the dissection of the underlying genetic components, using Quantitative Trait Locus (QTL) analysis and association mapping that may facilitate cultivar improvement through marker-assisted breeding. Cowpea accumulates iron and zinc in both grains and leaves, but fresh leaves have greater iron and zinc content than grains (Mamiro, 2011). The entire plant can be used for either human or livestock consumption, but in Brazil only the grains are utilized. Identifying QTLs/genes for iron/zinc storage and metabolism in cowpea can help in biofortification programs, especially by approaching the enhancement of iron and zinc storage in grains via expression of genes involved in iron/zinc storage and metabolism, since cowpea is not grown for leaf consumption in northeastern Brazil. In this study, we combined bioinformatics approaches to select potential genetic components of iron and zinc content in cowpea. Future perspectives are to use the candidate genes to test for tissue co-expression, for differential expression among lines with contrasting iron and zinc accumulation phenotype, and for SNP (Single Nucleotide Polymorphism) development and testing co-segregation with the metal content phenotypes, which could be applied as molecular marker for biofortification breeding purposes. Material and Methods The protein sequences used as prototype for the pathway reconstruction of iron and zinc homeostasis in cowpea were obtained from the NCBI non redundant protein data-bank after literature search. A brief description of each protein in the pathway along with the respective accession numbers are given in Table 1. The sequences were aligned against cowpea assembled ESTs through a tBLASTn search, adopting a cut-off e-value of 10e-05. ZIP- and Ferritin-matching ESTs were further analyzed by means of a perl script to assure that ESTs hitting both of the ZIP- and Ferritin-prototype proteins were assigned to the most similar one. The candidate sequences were aligned back to the proteins through BLASTx to check for frame orientation and frame-shift errors. Sequences presenting negative orientation were reverse complemented with the program revseq and those with frame-shifts were corrected using the program FRAMEDP. After correction, the sequences were subjected to conserved domain analysis utilizing the CDsearch tool from NCBI. Results A putative iron/zinc uptake, storage and metabolism pathway was successfully in silico reconstructed for cowpea root epidermis (Figure 1). 10 out of the 13 prototype proteins were already described in root epidermis of other plants, the exception are the human PCBP protein that chelates Fe 2+ ion and delivers it to Ferritins, and the chloroplastic Ferritin, which is not expected to be expressed in root epidermis. Cowpea presents variable number of matching EST for each prototype protein (Table 1). Not all candidate EST showed a complete coding sequence, as denoted by the number of complete conserved domain (Table 1). Discussion A putative pathway for uptake, storage and metabolism of iron and zinc in cowpea root epidermis is presented here. The pathway is an in silico attempt to select strong candidates for future phenotipic association, followed by functional studies. To become available for the plant, insoluble ferric oxides have to be first solubilized.

Dicotiledoneous plants affect the soluble iron content in the soil acidifying the rhizosphere by proton extrusion activity of the AHA protein family, rendering soluble Fe 3+, which is reduced to Fe2+ by the membrane reductase FRO2. Then, the bivalent iron is imported by the metal transporters IRT1, ZIP1 and NRAMP. This uptake system is dependent on the activity of FER transcription factor (Ivanov et al., 2012). In the in silico reconstructed pathway, 14 coding sequences were found for the AHA gene family, seven for FRO2, 13 for IRT1, one for ZIP1 and nine for NRAMP. Complete coding sequences were found only in the IRT subfamily. Proteins in this family are notably zinc/iron transporter but in vitro assays also showed binding affinity for copper, cadmium, cobalt, nickel and manganese (Morrissey and Guerinot, 2009); potentially toxic micronutrients for which no root epidermal transporters have yet been identified. The sequences here identified can serve for future functional studies to discover transporters for each of those micronutrients in natura. The sequence incompleteness observed may be due to low number of sequences available for the analysis, which was performed with 40.000 tentative contigs, assembled from 190.000 EST encompassing public and proprietary ESTs from the NordEST cowpea sequencing project. The same trend was observed for the other prototype proteins in the reconstructed pathway, except for the bHLH-containing FER transcription factor. bHLH proteins function as transcripional regulators of several pathways, from plant and organ development to secondary metabolism (CarreteroPaulet et al., 2010). Besides, bHLH are small proteins with around 300 amino acids. These two features could explain why more complete coding sequences were found for the FER transcription factor, despite the low sequence number for cowpea. Table 1: Proteins regulating Zinc and Iron homeostasis in the root apical zone. These proteins where used as prototypes for reconstruction of the pathway in Vigna unguiculata. For pathway details see figure 1. Family ZIP Description ZRT, IRT-like proteins involved in Zinc/Iron transport Natural resistance-associated macrophage protein involved in iron aquisition Vacuolar iron uptake transporter Yellow stripe-like/Oligopeptide transporter involved in Fe and other metal transport Cation diffusion facilitator translocates Zn2+ from cytosol into vacuoles Iron storage protein forming hollow sphere able to store up to 4500 iron atoms Iron chaperone delivering (Fe )3 to Ferritin in humans Ferric-chelate reduction oxidase, reducing Fe3+ to Fe2+ Polyeptide chelating Fe3+ probable involved in phloem-mediated long distance metal transport ATP-driven proton extrusion pump involved in soil acidification Fe-responsive bHLH transcription factor
2+

Gene Prototype AtZIP1 AtIRT1

Accession AEE75242 Q38856 AFQ37304 NP_178286 Q6R3L0 Q9ZT63 AAC06026 AAZ09196 NP_006187 Q9LMM2 CAC84735 NP_001190870 NP_001234654

Number of Matching Vigna EST(a) 1(0) 13(3) 9(0) 1(1) 9(0) 3(0) 17(5) 4(2) 18(6) 7(0) 4(4) 14(0) 30(29)

NRAMP VIT YSL CDF Ferritin

AhNRAMP AtVIT1 AtYSL1 AtZAT1 VuFer3 VuAOX1

PCBP FRO ITP AHA FER

HsPCBP1 AtFRO2 RcITP2 AtHA2 SlFER

(a) In parenthesis is the number of complete coding sequences, obtained through the analysis of conserved domains. Iron is also potentially toxic if reaching high intracellular concentration. To avoid Fe 2+ and Fe3+ reaching toxic levels, the ions can be chelated and transported to other plant parts or stored in the vacuoles until its allocation to other

plant parts. The tonoplastic CDF protein and vacuolar VIT protein transport zinc and iron to be stored in the vacuoles, while the vacuolar isoforms of NRAMP mobilizes vacuolar iron to the cytoplasm and the iron-transporting protein ITP chelates Fe3+ in plants and are involved in phloem-mediated long distance iron transfer, delivering iron to supply the demands of plant tissues and organs (Kruger et al., 2002; Kim et al., 2006; Gollhofer et al., 2011). Other chelating proteins are the ferritins that are able to store up to 4500 iron atoms. However, Fe 2+ loading of ferritin in human is dependent on the iron chaperone activity of the PCBP1 protein that delivers Fe2+ to ferritin (Shi et al., 2008). In our analysis we found 17 tentative contigs with homology to mitochondrial ferritins and four tentative contigs homologous to chloroplastic ferritins. Although the latter is not expected to be expressed in roots, including its prototype in the search strategy enable us to better discriminate the mitochondrial candidates. In plants, an iron chaperone with similar function such as the human PCBP1 is not known. However, we report the presence of six complete coding sequences similar to the human protein out of 18 tentative contigs found. If the plant homologs bind iron atoms or even if they have the same functionality must yet be researched.

Figure 1: Putative iron and zinc metabolism pathway reconstructed in silico for cowpea root epidermis. Dashed arrows indicate the genes under positive regulatory control of the bHLH FER transcription factor. Conclusion Biofortification of cowpea with Fe and Zn is a highly relevant issue in terms of designing new approach for increasing grain Fe and Zn and for selecting the most suitable parental lines in breeding programs aimed at improving grain metal content. Our findings may be seen as one first attempt to this goal, since it provides genetic components that may contribute to grain Fe and Zn by affecting the Fe soil availability, root uptake and phloem loading of Fe and Zn. The pathway elements can aid in finding gene isoforms, alleles and promoters for transgenic approaches, as well as for testing gene activity in the different lines. References Bouis, H. E. Micronutrient fortification of plants through breeding: can it improve nutrition in man at low cost? Proceedings of The Nutrion Society, v. 62, p. 403-411, 2004. Carvalho, L. C. B.; Damasceno-Silva, K. J.; Rocha, M. M.; Franco, L. J. D.; Almeida-Silva, L. R.; Carvalho, J. S.; Sousa, C. M. B.; Lustosa-Silva, J. D. Avaliao de populaes biofortificadas em feijo-caupi por meio de cruzamentos

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