The PI-SceI endonuclease from yeast belongs to a protein family whose members contain two conserved dodecapeptide motifs within their primary sequences. The function of two acidic residues within these motifs, Asp218 and Asp326, was examined by substituting alanine, asparagine, and glutamic acid residues at these positions. All of the purified mutant proteins bind to the PI-SceI recognition site with the same affinity and specificity as the wild-type enzyme. By contrast, substituting alanine or asparagine amino acids at the two positions completely eliminates strand cleavage of substrate DNA, whereas substitution with glutamic acid markedly reduces the cleavage activity. Experiments using nicked substrates demonstrate that the wild-type enzyme shows no strand preference during cleavage. These results are consistent with a model in which both acidic residues are part of a single catalytic center that cleaves both DNA strands. Furthermore, substrate binding by wild-type PI-SceI stimulates hydroxyl radical or hydroxide ion attack at the cleavage site while binding by the alanine-substituted proteins either stimulates this attack significantly less or protects the DNA at this position. These finding are discussed in terms of possible reaction mechanisms for PI-SceI-mediated endonucleolytic cleavage.

To understand how plants from the Fabaceae family maintain zinc (Zn) homeostasis, we have characterized the kinetics of three Zn transporting proteins from the ZIP family of divalent metal transporters in the model legume Medicago truncatula. Of six ZIP's studied, MtZIP1, MtZIP5 and MtZIP6 were the only members from this family determined to transport Zn and were further characterized. MtZIP1 has a low affinity for Zn with a K(m) of 1 μM as compared to MtZIP5 and MtZIP6 that have a higher affinity for Zn with K(m) of 0.4 μM and 0.3 μM, respectively. Zn transport by MtZIP1 was more sensitive to inhibition by copper (Cu) concentrations than MtZIP5 and MtZIP6, because 3 μM Cu inhibited Zn transport by 80% in MtZIP1 while 5 μM Cu was required to achieve the same inhibition of Zn transport in MtZIP5 and MtZIP6. Cadmium (Cd) had a greater effect on the ability of MtZIP1 to transport Zn than MtZIP5 and MtZIP6, because at a concentration of 3 μM Cd, the Zn transport by MtZIP1 was inhibited 55% and the transport of Zn by MtZIP5 and MtZIP6 was inhibited by 20-30%. However, only MtZIP6 transported Cd at higher rates than those observed in the control plasmid pFL61, demonstrating a low affinity for Cd based on a K(m) of 57 μM. These results suggest that Medicago truncatula has both high and low affinity Zn transporters to maintain Zn homeostasis and that these transporters may function in different compartments within the plant.

Snap beans (Phaseolus vulgaris L.) are a food source that can contribute to dietary Ca requirements in humans. Factors which might enhance the concentration of Ca in snap bean pods have been investigated by measuring whole-plant net Ca influx, whole-plant Ca partitioning, and various growth parameters in two snap bean cultivars—Hystyle and Labrador—that differ in pod Ca concentration. Plants were grown hydroponically under controlled environmental conditions while being provided adequate quantities of Ca. The concentration of Ca in pods (dry weight basis) was 52% higher in `Hystyle', relative to `Labrador', but net Ca influx throughout crop development or total plant Ca content at three stages of development were similar in both cultivars, demonstrating that pod Ca concentration differences were not due to differences in total plant Ca influx. However, `Hystyle' partitioned more total plant Ca to pods, relative to `Labrador'. Calcium flux analysis also revealed that daily rates of whole-plant net Ca influx gradually declined throughout the period of pod growth in both cultivars; this decline was not related to whole-plant water influx. These results suggest that enhancements in whole-plant net Ca influx during pod growth and/or enhancements in the xylem transport of absorbed Ca to developing pods could increase the Ca concentration of snap bean pods.