STUDY OF LOW PHYTIC ACID 1 LOCUS IN MAIZE

Phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate; InsP6) is ubiquitous in eukaryotic cells and constitutes the major storage form of phosphate in plant seeds (from 60% to 80%). During maturation it is accumulated in the protein storage vacuole in inclusions called globoids; the phosphate groups present in phytic acid (PA) are able to form phytate salts (phytin) binding important mineral cations. In mature maize kernels, 80% of PA is localized in the scutellum and the remaining 20% in the aleurone layer. The phosphorus stored as PA is remobilized during germination by phytase enzymes: these are also found in many microorganisms. Regarding the involvement of P in agricultural production and its sustainability, it has been estimated that nearly 50% of elemental P used yearly in global agricultural activities is accumulated in the PA. PA forming mixed salts with mineral cations is mainly excreted by monogastric animals and humans because they do not have phytase activity in their digestive systems. Considering that seeds are an important component of animal feed and human food, the limitations of phosphorus and micronutrients bioavailability imply a decrease in their nutritional value. Furthermore the undigested phosphorous contained in excreted phytin can contribute to water pollution (eutrophication). These negative effects have led to breeding programmes which have the aim of reducing the PA content in the seeds of several cultivated plants. The main way to reach this result by conventional breeding is the isolation of low phytic acid (lpa) mutations, capable of restraining the biosynthesis or the storage of PA in the seed; the increased P and mineral cation bioavailability in lpa seeds is confirmed by nutritional trials. In maize three low phytic acid mutants have been isolated: lpa1 and lpa2 by chemical mutagenesis, lpa3 by transposon tagging. Compared to the other mutations in maize, lpa1 exhibited the major reduction of PA in the seed, this comes with a proportional increase of free P without changing the total P content. Taking advantage of this property, lpa mutants can be recognized by the HIP (high inorganic phosphate) phenotype of the seeds. The Lpa1 gene encodes for ZmMRP4 (accession number EF586878) a multidrug-associated-protein (MRP) belonging to the subfamily of ATP-binding cassette (ABC) transmembrane transporters. MRP proteins are implicated in different roles like the transport of organic ions and anthocyanins, detoxification of xenobiotic compounds, transpiration control, and tolerance to oxidative stress. The role of this MRP protein is not completely understood but it is fundamental for phytic acid accumulation and viability of seeds. low phytic acid mutants isolated in rice and soybean are related to defects in homologues of the maize ABC transporter. It was observed that lpa mutations found in several crops usually bring pleiotropic effects on plant and seed performance, such as reduced germination and emergence rate, lower seed filling, weakening in stress resistance. The presence of pleiotropic effects shows that lpa mutations influence not only the seed but also the whole plant and its production. This can reflect the relevance of inositol phosphates as multifunctioning molecules, and their involvement in fundamental signaling and developmental pathways, like DNA repair, RNA editing, chromatin remodeling and control of gene expression. Furthermore phytic acid exhibits, by its ability to chelate iron, a potent antioxidant activity, avoiding the formation of reactive oxygen species. With the aim to isolate new maize low phytic acid mutants mutagenesis treatment were performed with EMS (ethyl-methanesulfonate). Since wild type mature maize seeds contain high amount of phytic phosphate and low free phosphate content, we screened the mutagenized population looking for seeds containing high levels of free phosphate (HIP phenotype), a typical feature of lpa. In previous studies a single recessive lpa mutation (originally named lpa241 and obtained by EMS pollen-tratment mutagenesis) was isolated and described, it was allelic to the lpa1-1 mutant, and was consequently renamed lpa1-241. A first evidence of non-Mendelian inheritance of lpa1 trait came from the appearance of unexpected free phosphate phenotypes in Lpa1/lpa1-241. When heterozygous families were selfed, we observed an overall increase of the mutant phenotype ratio due to the appearance of weak and intermediate phenotype, not consistent with a monogenic recessive mutation. This phenomenon can be explained with a partial Lpa1 allele silencing caused by trans interaction with the paramutagenic lpa1-241 allele. We performed 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. 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. We isolate a new maize (Zea mays L.) low phytic acid 1 mutant allele obtained by chemical EMS seed mutagenesis. We performed the allelism test with two other lpa1 mutants: lpa1-1 and lpa1-241, our mutant failed to complement these mutants. This mutant, named lpa1-7, exhibits a monogenic recessive inheritance and lethality as homozygous. We demonstrate that in vitro cultivation can overcome lethality allowing the growth of adult plants and we report data regarding embryo and leaf abnormalities and other defects caused by negative pleiotropic effects of this mutation. We conducted two experiments to ascertain the nature of lpa1-7 and. we also performed physiological analysis, histological observations and considerations regarding the effects of the lpa1 mutations on the plant. Pigmented maize contains anthocyanins and phenolic compounds which are phytochemicals synthesized in the plant by secondary metabolism; although these compounds are considered as non-nutritive, in these years the interest in antioxidant and bioactive properties has increased due to their health benefits. Anthocyanins are water soluble secondary metabolites belonging to the class of flavonoids and they play important roles in several aspect of plant biology. The anthocyanins are present in the vacuole in a glycosilated form and their colour is influenced in part by the pH of this compart. In maize they are synthesized by a complex pathway made up of more than 20 genes, and regulated by two classes of transcription factors: r1/b1 bHLH genes and c1/pl1/p1 MYB gene families. Our aim is the constitution of maize inbred lines carrying low phytic acid mutations together with regulatory genes pushing the anthocyanin accumulation in the kernels and seedlings, so they can compensate the leak in antioxidant activity induced by the low phytic acid mutation. We found that the lpa1-241 line is able to alter the accumulation of anthocyanin in kernel tissues. The anthocyanins, are present in the vacuole where their colour is dependent on the pH. In maize the anthocyanins are cytoplasmically synthesized molecules probably transported in the vacuole by ZmMRP3 gene activity. We observed an interaction between the accumulation of anthocyanin pigments in the kernel and the lpa mutations. In fact the lpa1-241 mutant accumulates a higher level of anthocyanins as compared to wild type either in the embryo or in the aleurone layer in a genotype able to accumulate anthocyanin. Furthermore, we demonstrate that these pigments are mislocalised in the cytoplasm, conferring a blue pigmentation of the scutellum, because of the neutral/basic pH of this cellular compartment; expression analysis showed a reduction of ZmMRP3 anthocyanins’ transporter gene expression. On the whole, these data strongly suggest a possible interaction between the lpa mutation and anthocyanin accumulation and compartmentalization in the kernel.