Elena Cassani

Phytic acid prevents oxidative stress in seeds: evidence from a maize (Zea mays L.) low phytic acid mutant

A maize mutant defective in the synthesis of phytic acid during seed maturation was used as a tool to study the consequences of the lack of this important reserve substance on seed survival. Data on germinability, free iron level, free radical relative abundance, protein carbonylation level, damage to DNA, degree of lipid peroxidation, alpha- and gamma-tocopherol amount and antioxidant capacity were recorded on seeds of maize B73 and of an isogenic low phytic acid mutant (lpa1-241), either unaged or incubated for 7 d in accelerated ageing conditions (46 degrees C and 100% relative humidity). The lpa1-241 mutant, compared to wild type (wt), showed a lower germination capacity, which decreased further after accelerated ageing. Whole lpa1-241 mutant kernels contained about 50% more free or weakly bound iron than wt ones and showed a higher content of free radicals, mainly concentrated in embryos; in addition, upon accelerated ageing, lpa1-241 seed proteins were more carbonylated and DNA was more damaged, whereas lipids did not appear to be more peroxidated, but the gamma-tocopherol content was decreased by about 50%. These findings can be interpreted in terms of previously reported but never proven antioxidant activity of phytic acid through iron complexation. Therefore, a novel role in plant seed physiology can be assigned to phytic acid, that is, protection against oxidative stress during the seeds life span. As in maize kernels the greater part of phytic acid (and thus of metal ions) is concentrated in the embryo, its antioxidant action may be of particular relevance in this crop.

A defective ABC transporter of the MRP family, responsible for the bean lpa1 mutation, affects the regulation of the phytic acid pathway, reduces seed myo‐inositol and alters ABA sensitivity

• 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.

A paramutation phenomenon is involved in the genetics of maize low phytic acid1-241 (lpa1-241) trait

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.

Characterization of the first dominant dwarf maize mutant carrying a single amino acid insertion in the VHYNP domain of the dwarf8 gene

The “green revolution” involving mainly wheat and rice was based on the use by breeders of semidominant mutations involved in the signal transduction pathway of Gibberellin (GA). In particular, mutations in the Reduced height (Rht) gene of wheat have been used to reduce plant height and consequently to avoid storm damage and lodging. These genes have been cloned and they encode for DELLA proteins which contain an N-terminal DELLA and a VHYNP domain essential for GA-dependent degradation of these proteins. In maize several mutations have been isolated which affect gibberellin biosynthesis and perception and in particular, mutations in Dwarf8 (D8) gene cause a severe dwarfing phenotype. D8 gene has been identified as an orthologue of Rht (Reduced height), Slr1(Slender rice 1) and Gibberellic Acid Insensitive (GAI) genes, this latter is a negative regulator of GA response in Arabidopsis. In this work, for the first time, we isolated and characterized a single amino acid insertion in the VHYNP domain of D8 maize gene causing the appearance of a dominant dwarf mutation. This spontaneous mutation, named D8-1023, showed a phenotype which is less severe in comparison with the other D8 mutants previously isolated which have modifications in the DELLA domain. This mutant appears to be an useful tool either to study the mechanism of GA-modulated growth in plants or to lower the height of maize tropical germplasm for breeding purposes.

Study of Low Phytic Acid1-7 (lpa1-7), a New ZmMRP4 Mutation in Maize

Phytic acid (PA), myo-inositol 1,2,3,4,5,6-hexakisphosphate, is the main storage form of phosphorus in plants. It is localized in seeds, deposited as mixed salts of mineral cations in protein storage vacuoles; during germination, it is hydrolyzed by phytases. When seeds are used as food/feed, PA and the bound cations are poorly bioavailable for human and monogastric livestock due to their lack of phytase activity. Reducing the amount of PA is one strategy to solve these problems and is an objective of genetic improvement for improving the nutritional properties of major crops. In this work, we present data on the isolation of a new maize (Zea mays L.) low phytic acid 1 (lpa1) mutant allele obtained by chemical mutagenesis. This mutant, named lpa1-7, is able to accumulate less phytic phosphorus and a higher level of free inorganic phosphate in the seeds compared with wild type. It 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.

The low phytic acid1-241 (lpa1-241) maize mutation alters the accumulation of anthocyanin pigment in the kernel

The lpa1 mutations in maize are caused by lesions in the ZmMRP4 (multidrug resistance-associated proteins 4) gene. In previous studies (Raboy et al. in Plant Physiol 124:355–368, 2000; Pilu et al. in Theor Appl Genet 107:980–987, 2003a; Shi et al. Nat Biotechnol 25:930–937, 2007), several mutations have been isolated in this locus causing a reduction of phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate, or InsP6) content and an equivalent increasing of free phosphate. In particular, the lpa1-241 mutation causes a reduction of up to 90% of phytic acid, associated with strong pleiotropic effects on the whole plant. In this work, we show, for the first time to our knowledge, 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 (about 3.8-fold) or in the aleurone layer (about 0.3-fold) 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. As a matter of fact, the propionate treatment, causing a specific acidification of the cytoplasm, restored the red pigmentation of the scutellum in the mutant and 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 compartmentalisation in the kernel.

Isolation and characterization of a new mutant allele of brachytic 2 maize gene

More than 40 monogenic dwarfing mutants have been described in maize; however, the majority of these lead to great reductions in grain yield and, consequently, they have not been used to enhance crop yield in germplasm that is sensible to lodging. An exception in terms of commercial value is the maize mutant brachytic 2 (br2). Br2 gene was cloned in 2003, and it encodes for a protein most probably involved in auxin polar transport. We have isolated a new brachytic mutation that is allelic to the br2 locus and denoted this novel mutant as br2–23. Characterization of this mutant revealed that the br2 mutation modified not only the length of the internodes, as previously reported, but the structure of the leaves as well. Br2–23/br2–23 heterozygotes have a useful intermediate phenotype in terms of plant height, ear height and leaf angle, suggesting a possible utilization of this effect in developing new hybrids. This mutant also appears to be an useful tool by which to study the switch points of the complex developmental program determining maize plant height and architecture.

Low Phytic Acid 1 Mutation in Maize Modifies Density, Starch Properties, Cations, and Fiber Contents in the Seed

Monogastric animals are unable to digest phytic acid, so it represents an antinutritional factor and also an environmental problem. One strategy to solve this problem is the utilization of low phytic acid (lpa) mutants that accumulate low levels of phytic P and high levels of free phosphate in the seeds; among the lpa maize mutants lpa1 exhibited the highest reduction of phytic acid in the seed. This study indicated that the low phytic acid mutations exerted pleiotropic effects not directly connected to the phytic acid pathway, such as on seed density, content of ions, and the antioxidant compounds present in the kernels. Furthermore some nutritional properties of the flour were altered by the lpa1 mutations, in particular lignin and protein content, while the starch does not seem to be modified as to the total amount and in the amylose/amylopectin ratio, but alterations were noticed in the structure and size of granules.

Effect of flavonoid pigments on the accumulation of fumonisin B1 in the maize kernel

Mycotoxins are secondary metabolites with potential dangers for animal and human health. In particular, maize (Zea mays L.) infection caused by Fusarium and the consequent fumonisin contamination is widespread in several countries such as Italy. We developed six maize populations differing in their constitution of regulatory genes able to accumulate respectively anthocyanins in the aleurone layer (r1 gene), pericarp (b1 and pl1 genes) and phlobaphene in the pericarp (p1 gene). These coloured populations, with the related control colourless populations were analysed for mycotoxin content in the kernels during three field seasons with the aim of understanding if there were any correlations with their ability to accumulate flavonoids in kernel tissues. Our results indicate that accumulation of flavonoid pigments in the seeds, in particular phlobaphenes, is able to reduce the level of fumonisin B1. This finding could be used to minimize kernel mycotoxin contamination in this crop, in particular, the development of sweet, pop and polenta coloured corn varieties will help the farmer to keep the level of fumonisin under the threshold of contamination established for human corn consumption.

Analysis of chromosome number and speculations on the origin of Arundo donax L. (Giant Reed)

Arundo donax (commonly called Giant Reed) is a perennial rhizomatous grass native to Asia, nowadays diffused all over the world. Due to its high biomass production and great adaptability to marginal land, interest in this species is increasing. In fact A. donax could represent an important and promising energy crop for heat and bioethanol second generation production. The propagation of A. donax is strictly agamic by rhizome fragmentation and cane node germination, strongly limiting the possibility of genetic improvement by breeding. The sterility could be caused by the fact that A. donax is a hybrid with uneven ploidy or a triploid species. It is difficult to propose an explanation for its sterility, because the chromosome number of A. donax is still a matter of debate, due to the high number and small size of the chromosomes; in the bibliography different counts ranging from 40 to 110 are reported. With the aim of establishing the chromosome number of A. donax we selected and counted 17 metaphase plates prepared from root tips obtained by hydroponic cultivation of cane nodes; our counts showed that A. donax most probably has 110 chromosomes. Our results suggested us two possible hypotheses, also based on SSR molecular marker results, concerning the evolutionary processes involved in the origins of A. Donax.