DIRECT MEASUREMENT OF FE-59-LABELED FE2+ INFLUX IN ROOTS OF PEA USINGA CHELATOR BUFFER SYSTEM TO CONTROL FREE FE2+ IN SOLUTION

Fe2+ transport in plants has been difficult to quantify because of the inability to control Fe2+ activity in aerated solutions and nonspecif ic binding of Fe to cell walls. In this study, a idyl)-5,6-diphenyl-1, 2,4-triazine-4'4''-disulfonic acid buffer system was used to control f ree Fe2+ in uptake solutions. Additionally, desorption methodologies w ere developed to adequately remove nonspecifically bound Fe from the r oot apoplasm. This enabled us to quantify unidirectional Fe2+ influx v ia radiotracer (Fe-59) uptake in roots of pea (Pisum sativum cv Sparkl e) and its single gene mutant brz, an Fe hyperaccumulator. Fe influx i nto roots was dramatically inhibited by low temperature, indicating th at the measured Fe accumulation in these roots was due to true influx across the plasma membrane rather than nonspecific binding to the root apoplasm. Both Fe2+ influx and Fe translocation to the shoots were st imulated by Fe deficiency in Sparkle. Additionally, brz, a mutant that constitutively exhibits high ferric reductase activity, exhibited hig her Fe2+ influx rates than +Fe-grown Sparkle. These results suggest th at either Fe deficiency triggers the induction of the Fe2+ transporter or that the enhanced ferric reductase activity somehow stimulates the activity of the existing Fe2+ transport protein.