Overexpression of METAL TOLERANCE PROTEIN8 reveals new aspects of metal transport in Arabidopsis thaliana seeds.

METAL TOLERANCE PROTEIN8 (MTP8) of Arabidopsis thaliana is a member of the CATION DIFFUSION FACILITATOR (CDF) family of proteins that transports primarily manganese (Mn), but also iron (Fe). MTP8 mediates Mn allocation to specific cell types in the developing embryo, and Fe re-allocation as well as Mn tolerance during imbibition. We analysed if an overexpression of MTP8 driven by the CaMV 35S promoter has an effect on Mn tolerance during imbibition and on Mn and Fe storage in seeds, which would render it a biofortification target. Fe, Mn and Zn concentrations in MTP8-overexpressing lines in wild type and vit1-1 backgrounds were analysed by ICP-MS. Distribution of metals in intact seeds was determined by synchrotron µXRF tomography. MTP8 overexpression led to a strongly increased Mn tolerance of seeds during imbibition, supporting its effectiveness in loading excess Mn into the vacuole. In mature seeds, MTP8 overexpression did not cause a consistent increase in Mn and Fe accumulation, and it did not change the allocation pattern of these metals. Zn concentrations were consistently increased in bulk samples. The results demonstrate that Mn and Fe allocation is not determined primarily by the MTP8 expression pattern, suggesting either a cell type-specific provision of metals for vacuolar sequestration by upstream transport processes, or the determination of MTP8 activity by post-translational regulation.

The nuclear protein Poly(ADP-ribose) polymerase 3 (AtPARP3) is required for seed storability in Arabidopsis thaliana.

The deterioration of seeds during prolonged storage results in a reduction of viability and germination rate. DNA damage is one of the major cellular defects associated with seed deterioration. It is provoked by the formation of reactive oxygen species (ROS) even in the quiescent state of the desiccated seed. In contrast to other stages of seed life, DNA repair during storage is hindered through the low seed water content; thereby DNA lesions can accumulate. To allow subsequent seedling development, DNA repair has thus to be initiated immediately upon imbibition. Poly(ADP-ribose) polymerases (PARPs) are important components in the DNA damage response in humans. Arabidopsis thaliana contains three homologues to the human HsPARP1 protein. Of these three, only AtPARP3 was very highly expressed in seeds. Histochemical GUS staining of embryos and endosperm layers revealed strong promoter activity of AtPARP3 during all steps of germination. This coincided with high ROS activity and indicated a role of the nuclear-localised AtPARP3 in DNA repair during germination. Accordingly, stored parp3-1 mutant seeds lacking AtPARP3 expression displayed a delay in germination as compared to Col-0 wild-type seeds. A controlled deterioration test showed that the mutant seeds were hypersensitive to unfavourable storage conditions. The results demonstrate that AtPARP3 is an important component of seed storability and viability.