Phytic acid, myo-inositol 1,2,3,4,5,6-hexakisphosphate, is the major storage compound of phosphorous (P) in plants, predominantly accumulating in seeds (up to 4-5% of dry weight) and pollen. In cereals, phytic acid is deposited in embryo and aleurone grain tissues as a mixed "phytate" salt of potassium and magnesium, although phytates contain other mineral cations such as iron and zinc. During germination, phytates are broken down by the action of phytases, releasing their P, minerals and myo-inositol which become available to the growing seedling. Phytic acid represents an anti-nutritional factor for animals, and isolation of maize low phytic acid ( lpa) mutants provides a novel approach to study its biochemical pathway and to tackle the nutritional problems associated with it. Following chemical mutagenesis of pollen, we have isolated a viable recessive mutant named lpa 241 showing about 90% reduction of phytic acid and about a tenfold increase in seed-free phosphate content. Although germination rate was decreased by about 30% compared to wild-type, developement of mutant plants was apparentely unaffected. The results of the genetic, biochemical and molecular characterization experiments carried out by SSR mapping, MDD-HPLC and RT-PCR are consistent with a mutation affecting the MIPS1S gene, coding for the first enzyme of the phytic acid biosynthetic pathway.

Key words: inositol 1,4,5-tris-phosphate kinase, low phytic acid, myo-inositol-1,2,3,4,5,6-hexakisphosphate (InsP 6 ), myo-inositol-phosphate monophosphatase (IMP), myo-inositol-3-phosphate synthase (MIPS), multidrug resistance protein (MRP) type ATP-binding cassette (ABC) transporter, Phaseolus vulgaris (common bean). Summary• We previously identified the lpa1 (low phytic acid) 280-10 line that carries a mutation conferring a 90% reduction in phytic acid (InsP 6 ) content. In contrast to other lpa mutants, lpa1(280-10) does not display negative pleiotropic effects. In the present paper, we have identified the mutated gene and analysed its impact on the phytic acid pathway.• Here, we mapped the lpa1(280-10) mutation by bulk analysis on a segregating F 2 population, an then, by comparison with the soybean genome, we identified and sequenced a candidate gene. The InsP 6 pathway was analysed by gene expression and quantification of metabolites.• The mutated Pvmrp1(280-10) cosegregates with the lpa1(280-10) mutation, and the expression level of several genes of the InsP 6 pathway are reduced in the lpa1(280-10) mutant as well as the inositol and raffinosaccharide content. PvMrp2, a very similar paralogue of PvMrp1 was also mapped and sequenced.• The lpa1 mutation in beans is likely the result of a defective Mrp1 gene (orthologous to the lpa genes AtMRP5 and ZmMRP4), while its Mrp2 paralog is not able to complement the mutant phenotype in the seed. This mutation appears to down-regulate the InsP 6 pathway at the transcriptional level, as well as altering inositol-related metabolism and affecting ABA sensitivity.Abbreviations: ABC, ATP-binding cassette; MRP, multidrug resistance-associated protein; InsP 6, myo-inositol-1,2,3,4,5,6-hexakisphosphate; IMP, myo-inositolphosphate monophosphatase; MIPS, myo-inositol-3-phosphate synthase; MIK, myo-inositol kinase; IPK2, inositol 1,4,5-tris-phosphate kinase; ITPK, inositol 1,3,4-triphosphate 5 ⁄ 6-kinase; IPK1, inositol 1,3,4,5,6 pentakisphosphate 2-kinase.

Plasma membrane-localized pattern recognition receptors (PRRs) such as FLAGELLIN SENSING2 (FLS2), EF-TU RECEPTOR (EFR), and CHITIN ELICITOR RECEPTOR KINASE1 (CERK1) recognize microbe-associated molecular patterns (MAMPs) to activate pattern-triggered immunity (PTI). A reverse genetics approach on genes responsive to the priming agent b-aminobutyric acid (BABA) revealed IMPAIRED OOMYCETE SUSCEPTIBILITY1 (IOS1) as a critical PTI player. Arabidopsis thaliana ios1 mutants were hypersusceptible to Pseudomonas syringae bacteria. Accordingly, ios1 mutants showed defective PTI responses, notably delayed upregulation of the PTI marker gene FLG22-INDUCED RECEPTOR-LIKE KINASE1, reduced callose deposition, and mitogen-activated protein kinase activation upon MAMP treatment. Moreover, Arabidopsis lines overexpressing IOS1 were more resistant to bacteria and showed a primed PTI response. In vitro pull-down, bimolecular fluorescence complementation, coimmunoprecipitation, and mass spectrometry analyses supported the existence of complexes between the membrane-localized IOS1 and BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1)-dependent PRRs FLS2 and EFR, as well as with the BAK1-independent PRR CERK1. IOS1 also associated with BAK1 in a ligand-independent manner and positively regulated FLS2-BAK1 complex formation upon MAMP treatment. In addition, IOS1 was critical for chitinmediated PTI. Finally, ios1 mutants were defective in BABA-induced resistance and priming. This work reveals IOS1 as a novel regulatory protein of FLS2-, EFR-, and CERK1-mediated signaling pathways that primes PTI activation. INTRODUCTIONPlants possess multilayered recognition systems that detect pathogens at various stages of infection and proliferation. Recognition of microbial invasion is essentially based upon the host's ability to distinguish between self and non-self components. Early microbial pathogens detection is performed by cell surfacelocalized pattern recognition receptors (PRRs) that sense pathogen-or microbe-associated molecular patterns (PAMPs or MAMPs) (Monaghan and Zipfel, 2012). Major examples of MAMPs are lipopolysaccharides present in the envelope of Gram-negative bacteria, eubacterial flagellin, eubacterial elongation factor Tu (EF-Tu), peptidoglycans from Gram-positive bacteria, methylated bacterial DNA fragments, and fungal cell wall-derived chitins (Girardin et al., 2002;Cook et al., 2004;Boller and Felix, 2009). MAMP recognition promptly triggers the activation of patterntriggered immunity (PTI) (Tsuda and Katagiri, 2010). Early PTI responses, such as calcium influx, production of reactive oxygen species (ROS), and activation of mitogen-activated protein (MAP) kinases, induce transcriptional reprogramming mediated by plant WRKY transcription factors as well as calmodulin binding proteins (Boller and Felix, 2009;Tena et al., 2011). In addition, Arabidopsis thaliana plants close stomata in a MAMP-dependent manner when in contact with bacteria (Melotto et al., 2006;Singh et al., 2012). Callose deposition and PTI marker gene up...

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So far, in maize, three classes of mutants involved in phytic acid biosynthesis have been isolated: lpa1, lpa2 and lpa3. In 2007, a gene tagging experiment performed by Shi et al. found that mutations in ZmMRP4 (multidrug resistance-associated proteins 4) gene cause lpa1 phenotype. In previous studies, we isolated and described a single recessive lpa mutation (originally named lpa241), which was allelic to the lpa1-1 mutant, and was consequently renamed lpa1-241. It showed a decrease in the expression of the myo-inositol (Ins)-3-phosphate synthase gene (mips1S). In this study, we present genetic and molecular analyses of the lpa1-241 mutation that indicate an epigenetic origin of this trait, that is, a paramutagenic interaction that results in meiotically heritable changes in ZmMRP4 gene expression, causing a strong pleiotropic effect on the whole plant. The use of a 5-Azacytidine treatment provided data suggesting an association between gene methylation and the lpa1-241 phenotype. To our knowledge, this is the first report of a paramutagenic activity not involving flavonoid biosynthesis in maize, but regarding a key enzyme of an important metabolic pathway in plants.