Phytic acid (myo-inositol-hexa-kis-phosphate, InsP6) is the main storage form for phosphate in seeds and in the 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 lpa-1 mutant, isolated in our lab, shows a point mutation in the 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 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 the 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 common bean wild type plants showed that both genes are similarly expressed in different organs with the exception of seeds, where only PvMRP1 is expressed. In order to analyze promoters of these two genes, constructs harboring portions of 1.5 kb of their putative sequences fused upstream of the GUS reporter were generated and used to transform two species: Arabidopsis thaliana and Medicago truncatula. In Arabidopsis plants high GUS activity was detected only in vascular tissue of different organs for both constructs. These results were in agreement with the qRT-PCR data showing expression of both genes at low level in leaf. However, surprisingly, plants harboring the construct with the PvMRP1 gene promoter did not show any GUS staining in the seed, as expected from PvMRP1 transcript profile in common bean. In order to understand if this discrepancy is due to differences between the common bean and the Arabidopsis seed, we decided to use the same approach in the model legume M. truncatula. Molecular and biochemical characterizations of transgenic M. truncatula plant lines are currently under way, and we are producing the T1 generation of M. truncatula transformants.
GENE REGULATION OF PHYTIC ACID TRANSPORTERS MAY EXPLAIN THE ABSENCE OF PLEIOTROPIC EFFECTS OF THE lpa-1 MUTATION IN Phaseolus vulgaris
COMINELLI E;SPARVOLI F;
2015
Abstract
Phytic acid (myo-inositol-hexa-kis-phosphate, InsP6) is the main storage form for phosphate in seeds and in the 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 lpa-1 mutant, isolated in our lab, shows a point mutation in the 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 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 the 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 common bean wild type plants showed that both genes are similarly expressed in different organs with the exception of seeds, where only PvMRP1 is expressed. In order to analyze promoters of these two genes, constructs harboring portions of 1.5 kb of their putative sequences fused upstream of the GUS reporter were generated and used to transform two species: Arabidopsis thaliana and Medicago truncatula. In Arabidopsis plants high GUS activity was detected only in vascular tissue of different organs for both constructs. These results were in agreement with the qRT-PCR data showing expression of both genes at low level in leaf. However, surprisingly, plants harboring the construct with the PvMRP1 gene promoter did not show any GUS staining in the seed, as expected from PvMRP1 transcript profile in common bean. In order to understand if this discrepancy is due to differences between the common bean and the Arabidopsis seed, we decided to use the same approach in the model legume M. truncatula. Molecular and biochemical characterizations of transgenic M. truncatula plant lines are currently under way, and we are producing the T1 generation of M. truncatula transformants.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
