Isolation and characterization of a new low phytic acid mutant in the common bean PvMRP1 gene and study of PvMRPs promoters in two different plant systems

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 pleiotropic effects, such as compromised germination and emergence, lower stress tolerance and poor seed filling. We previously isolated the common bean lpa1-1 mutant, affected in the PvMRP1 gene, coding for a putative tonoplastic phytic acid transporter (Panzeri et al., 2011). With the aim to isolate new common bean lpa mutants we partially screened an EMS population (ca. 700 among the total 1700 M4 lines; Porch et al., 2009) by the molybdate staining assay that was able to recognize the high inorganic phosphate (HIP) phenotype, a typical feature of lpa mutants. We identified a new lpa mutant, hereinafter called lpa1-2, as from allelism test with lpa1-1 mutant and sequence analyses, resulted to affect the PvMRP1 gene, as well. The lpa1-2 mutation consists in a premature stop codon. In this work, we present preliminary data of the phenotypic characterization of lpa1-2 mutant. The lpa1-1 and lpa1-2 mutant plants do not display negative pleiotropic effects and agronomic defects, as observed in other species affected in the orthologous genes. In common bean there is also the 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. 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 PvMRP1 is expressed at higher levels than PvMRP2 gene. 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 both plant species, 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, Arabidopsis 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. Probably this discrepancy is due to differences between the common bean and the Arabidopsis seed, as in the model legume Medicago truncatula PvMRP1 promoter drives GUS expression also in the seed.