Phytic acid (myo-inositol-hexa-kis-phosphate, InsP6) is the main storage form for phosphate in cereal and legume seeds and in plant it plays an important role in signal transduction in response to environmental stress and hormonal changes. InsP6 has a strong negative charge, able to chelate mono and divalent cations, such as iron, zinc, magnesium and calcium, essential minerals in the diet, reducing their bioavailabilty. Hence it is considered an anti-nutritional factor. Therefore the isolation of low phytic acid (lpa) mutants is considered a highly desirable objective in the genetic improvement of crops nutritional quality. However lpa mutants are often associated with negative effects such as compromised germination and emergence, lower stress tolerance and poor seed filling. The Phaseolus vulgaris lpa-1 mutant, isolated in our lab, shows a point mutation in PvMRP1 gene, ortholog of the Arabidopsis thaliana AtMRP5 gene coding for an ATP-Binding Cassette phytic acid transporter, located on the tonoplast. The lpa-1 mutant confers to seeds a 90% reduction of phytic acid content, a 25% reduction in raffinosaccharide content and a seven fold increase of free iron cations. The mutant plant does not display evdent pleiotropic effects and agronomic defects. In common bean there is also PvMRP2 gene, paralog of PvMRP1, probably able to complement the lpa-1 phenotype in other tissues than the seed, thus explaining the lack of pleiotropic effects in the bean mutant. Aim of this work is to study the PvMRP1 and PvMRP2 genes regulation. qRT-PCR analysis in wild type plants showed that both genes are similarly expressed in different organs with the exception of seeds, where only PvMRP1 is expressed. Some differences of the expression level of the two genes were observed in mutant plants. More detailed information on temporal and spatial expression of PvMRPs and key phytic acid biosynthetic genes (PvMIPSs, PvIMP, PvIPK2, PvIPK1) during common bean seed development in wild type and lpa-1 genotypes were obtained through in situ hybridization analysis, still in progress for some genes. The following compartments of the seed have been investigated during Phaseolus vulgaris seed development (early, middle and later stages): the integument of the seed with the function of protection and coating; the extra-embryonic tissues with trophic function (nucella and endothelium); reserve tissues and organs (endosperm and cotyledons); finally the embryo in two specific areas, apex meristem and cotyledonary leaves. The PvMRPs genes show some similarities in gene expression pattern in embryo and cotyledons. At early stages both genes are expressed in the entire embryo in wild type, while only in the embryo supporter in the mutant. However at later stages PvMRP1 is expressed in the cotyledons, while PvMRP2 is not. This pattern confirms the non-redundant function of the two genes in the main phytic acid storage compartment of common bean, already highlighted by the phenotype of the lpa-1 mutant seeds. Concerning the expression of biosynthetic genes in wild type genotype PvMIPSs gave similar hybridization signal as PvMRPs genes mainly at the globular stage, while in the lpa-1 seeds it is not expressed in the nucella. This detailed analysis of the expression of phytic acid biosynthesis and transport genes will help in elucidating the complex interaction between different seed compartments occurring during the synthesis and subsequent accumulation of InsP6 in developing seeds of common bean.
Phytic acid biosynthesis and transport: temporal and spatial gene expression analysis during Phaseolus vulgaris seed development
LOSA A;COMINELLI E;DAMINATI M G;BOLLINI R;SPARVOLI F
2015
Abstract
Phytic acid (myo-inositol-hexa-kis-phosphate, InsP6) is the main storage form for phosphate in cereal and legume seeds and in plant it plays an important role in signal transduction in response to environmental stress and hormonal changes. InsP6 has a strong negative charge, able to chelate mono and divalent cations, such as iron, zinc, magnesium and calcium, essential minerals in the diet, reducing their bioavailabilty. Hence it is considered an anti-nutritional factor. Therefore the isolation of low phytic acid (lpa) mutants is considered a highly desirable objective in the genetic improvement of crops nutritional quality. However lpa mutants are often associated with negative effects such as compromised germination and emergence, lower stress tolerance and poor seed filling. The Phaseolus vulgaris lpa-1 mutant, isolated in our lab, shows a point mutation in PvMRP1 gene, ortholog of the Arabidopsis thaliana AtMRP5 gene coding for an ATP-Binding Cassette phytic acid transporter, located on the tonoplast. The lpa-1 mutant confers to seeds a 90% reduction of phytic acid content, a 25% reduction in raffinosaccharide content and a seven fold increase of free iron cations. The mutant plant does not display evdent pleiotropic effects and agronomic defects. In common bean there is also PvMRP2 gene, paralog of PvMRP1, probably able to complement the lpa-1 phenotype in other tissues than the seed, thus explaining the lack of pleiotropic effects in the bean mutant. Aim of this work is to study the PvMRP1 and PvMRP2 genes regulation. qRT-PCR analysis in wild type plants showed that both genes are similarly expressed in different organs with the exception of seeds, where only PvMRP1 is expressed. Some differences of the expression level of the two genes were observed in mutant plants. More detailed information on temporal and spatial expression of PvMRPs and key phytic acid biosynthetic genes (PvMIPSs, PvIMP, PvIPK2, PvIPK1) during common bean seed development in wild type and lpa-1 genotypes were obtained through in situ hybridization analysis, still in progress for some genes. The following compartments of the seed have been investigated during Phaseolus vulgaris seed development (early, middle and later stages): the integument of the seed with the function of protection and coating; the extra-embryonic tissues with trophic function (nucella and endothelium); reserve tissues and organs (endosperm and cotyledons); finally the embryo in two specific areas, apex meristem and cotyledonary leaves. The PvMRPs genes show some similarities in gene expression pattern in embryo and cotyledons. At early stages both genes are expressed in the entire embryo in wild type, while only in the embryo supporter in the mutant. However at later stages PvMRP1 is expressed in the cotyledons, while PvMRP2 is not. This pattern confirms the non-redundant function of the two genes in the main phytic acid storage compartment of common bean, already highlighted by the phenotype of the lpa-1 mutant seeds. Concerning the expression of biosynthetic genes in wild type genotype PvMIPSs gave similar hybridization signal as PvMRPs genes mainly at the globular stage, while in the lpa-1 seeds it is not expressed in the nucella. This detailed analysis of the expression of phytic acid biosynthesis and transport genes will help in elucidating the complex interaction between different seed compartments occurring during the synthesis and subsequent accumulation of InsP6 in developing seeds of common bean.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
