Contents lists available at ScienceDirect Plant Science journal homepage: www.elsevier.com/locate/plantsci Phytic acid transport in Phaseolus vulgaris: A new low phytic acid mutant in the PvMRP1 gene and study of the PvMRPs promoters in two different plant systems Eleonora Cominelli a, ⁎ , Massimo Confalonieri b , Martina Carlessi a,c , Gaia Cortinovis a , Maria Gloria Daminati a , Timothy G. Porch d , Alessia Losa a,e , Francesca Sparvoli a a CNR – National Research Council, Institute of Agricultural Biology and Biotechnology (IBBA, CNR), Via E. Bassini, 15, 20133, Milan, Italy b CREA Research Centre for Animal Production and Aquaculture (CREA-ZA), Viale Piacenza 29, 26900, Lodi, Italy c Present address: Plantlab, Institute of Life Sciences, Scuola Superiore Sant’Anna, Via G. Guidiccioni, 8-10, 56010 Ghezzano (Pisa), Italy d USDA-ARS, Tropical Agriculture Research Station, 2200 P.A. Campos Avenue, Suite 201, Mayaguez, 00680, Puerto Rico e CREA Research Centre for Genomics and Bioinformatics (CREA-GB), Via Paullese 28, 26836 Montanaso Lombardo, Lodi, Italy ARTICLE INFO Keywords: Phytic acid low phytic acid (lpa) Multidrug resistance protein (MRP) type ATP- binding cassette (ABC) transporter Phaseolus vulgaris ABSTRACT Phytic acid (InsP 6 ) is the main storage form of phosphate in seeds. In the plant it plays an important role in response to environmental stress and hormonal changes. InsP 6 is a strong chelator of cations, reducing the bioavailability of essential minerals in the diet. Only a common bean low phytic acid (lpa1) mutant, affected in the PvMRP1 gene, coding for a putative tonoplastic phytic acid transporter, was described so far. This mutant is devoid of negative pleiotropic effects normally characterising lpa mutants. With the aim of isolating new common bean lpa mutants, an ethyl methane sulfonate mutagenized population was screened, resulting in the identification of an additional lpa1 allele. Other putative lpa lines were also isolated. The PvMRP2 gene is probably able to complement the phenotype of mutants affected in the PvMRP1 gene in tissues other than the seed. Only the PvMRP1 gene is expressed at appreciable levels in cotyledons. Arabidopsis thaliana and Medicago truncatula transgenic plants harbouring 1.5 kb portions of the intergenic 5′ sequences of both PvMRP genes, fused upstream of the GUS reporter, were generated. GUS activity in different organs suggests a refined, species- specific mechanisms of regulation of gene expression for these two PvMRP genes. 1. Introduction In the seed, up to 85% of total phosphorus is stored as phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate; InsP 6 ). During germina- tion, the activity of phytases remobilizes the phosphorus stored as InsP 6 to support seedling growth. As InsP 6 is highly negatively charged at physiological pH, it binds mineral cations and easily precipitates in the form of phytate salts. Humans and non-ruminants digest InsP 6 poorly because phytases are not present in their digestive tract. Consequently, the presence of InsP 6 limits mineral and phosphorus bioavailability and this compound is considered an anti-nutritional factor as well as a major problem for water eutrophication due to its presence in animal manures [1]. Recently it has become clear that InsP 6 , and its precursors and pirophosphorylated forms, derived from its metabolism (InsP 7 , InsP 8 ), play a key role in different plant cell processes, such as the regulation of hormone activity [2–4,5], abiotic and biotic stress response [6,7], cal- cium and sugar signalling [8,9], phosphorus homeostasis [10,11], photomorphogenesis [12], chromatin modification and remodeling [13] and mRNA nuclear export [14]. Two different InsP 6 biosynthetic pathways can be distinguished: 1) the lipid-independent one, predominant in the seeds, consisting in the sequential phosphorylation of myo-inositol (Ins) ring and soluble in- ositol phosphates (InsP s ); and 2) the lipid-dependent one, mainly active in vegetative tissues, where phosphatidylinositol (PtdIns) and PtdIns phosphates are the substrates of phosphorylation [15]. In both routes, an early or substrate supply phase and an early-intermediate phase that generate InsP 3 are present, then the two pathways converge in a late https://doi.org/10.1016/j.plantsci.2018.02.003 Received 22 December 2017; Received in revised form 2 February 2018; Accepted 5 February 2018 ⁎ Corresponding author at: CNR – National Research Council, Institute of Agricultural Biology and Biotechnology (IBBA, CNR), Via E. Bassini, 15, 20133, Milan, Italy. E-mail addresses: cominelli@ibba.cnr.it (E. Cominelli), massimo.confalonieri@crea.gov.it (M. Confalonieri), m.carlessi@santannapisa.it (M. Carlessi), gaia.cortinovis@studenti.unimi.it (G. Cortinovis), daminati@ibba.cnr.it (M.G. Daminati), Timothy.Porch@ARS.USDA.GOV (T.G. Porch), alessia.losa@crea.gov.it (A. Losa), sparvoli@ibba.cnr.it (F. Sparvoli). Abbreviations: lpa, low phytic acid; Ins, myo-inositol; InsP s , inositol phosphates; InsP 6 , phytic acid; PtdIns, phosphatidylinositol; MRP, multidrug resistance protein; ABC, ATP-binding cassette; EMS, ethyl methane sulfonate; GUS, β-glucuronidase; P, phosphorous; P i , inorganic phosphorous; HIP, high inorganic phosphorous; PAP, phytic acid phosphorous Plant Science 270 (2018) 1–12 Available online 07 February 2018 0168-9452/ © 2018 Elsevier B.V. All rights reserved. T