Phytic acid (myo-inositol-hexa-kis-phosphate, InsP6) is the main storage form for phosphate in cereals and legumes 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 the 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 lpa1 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 lpa1 mutant confers a 90% reduction of phytic acid content in seed, a 25% reduction in raffinosaccharide content and a seven fold increase of free iron cations in the seeds. The mutant plant does not display pleiotropic effects and agronomic defects. In common bean there is also PvMRP2 gene, paralog of PvMRP1, probably able to complement the lpa1 phenotype in other tissues than the seed, thus explaining the lack of pleiotropic effects in the bean mutant. The aim of this work is to study the PvMRP1 and PvMRP2 genes regulation. qRT-PCR analysis in wt 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 PvMRP genes expression during common bean seed development in wt and lpa1 is being obtained through in situ hybridization analysis. We are also interested to compare the expression of key genes involved in phytic acid biosynthesis, such as myo-inositol 3-phophate synthase (PvMIPSs), inositol mono-phosphatase (PvIMP), inositol 1,4,5-tris-phosphate kinase (PvIPK2) and inositol 1,3,4,5,6 pentakisphosphate 2-kinase (PvIPK1) in wt and lpa1 backgrounds. Moreover Arabidopsis transgenic plants, harboring a portion of 1.5 kb of putative promoters of PvMRP1 and PvMRP2 fused upstream of the GUS reporter gene revealed high GUS activity in vascular tissue of different plant organs for both constructs. These data disagree with the PvMRP1 gene expression in the seed, therefore a longer region upstream of the PvMRP1 and PvMRP2 genes will be analyzed, in order to identify their complete promoter regions.
Temporal and spatial expression analysis of genes involved in phytic acid (InsP6) biosynthesis and transport during Phaseolus vulgaris seed development
Losa A;Cominelli E;Daminati M G;Bollini R;Sparvoli F
2014
Abstract
Phytic acid (myo-inositol-hexa-kis-phosphate, InsP6) is the main storage form for phosphate in cereals and legumes 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 the 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 lpa1 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 lpa1 mutant confers a 90% reduction of phytic acid content in seed, a 25% reduction in raffinosaccharide content and a seven fold increase of free iron cations in the seeds. The mutant plant does not display pleiotropic effects and agronomic defects. In common bean there is also PvMRP2 gene, paralog of PvMRP1, probably able to complement the lpa1 phenotype in other tissues than the seed, thus explaining the lack of pleiotropic effects in the bean mutant. The aim of this work is to study the PvMRP1 and PvMRP2 genes regulation. qRT-PCR analysis in wt 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 PvMRP genes expression during common bean seed development in wt and lpa1 is being obtained through in situ hybridization analysis. We are also interested to compare the expression of key genes involved in phytic acid biosynthesis, such as myo-inositol 3-phophate synthase (PvMIPSs), inositol mono-phosphatase (PvIMP), inositol 1,4,5-tris-phosphate kinase (PvIPK2) and inositol 1,3,4,5,6 pentakisphosphate 2-kinase (PvIPK1) in wt and lpa1 backgrounds. Moreover Arabidopsis transgenic plants, harboring a portion of 1.5 kb of putative promoters of PvMRP1 and PvMRP2 fused upstream of the GUS reporter gene revealed high GUS activity in vascular tissue of different plant organs for both constructs. These data disagree with the PvMRP1 gene expression in the seed, therefore a longer region upstream of the PvMRP1 and PvMRP2 genes will be analyzed, in order to identify their complete promoter regions.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
