DIFFERENTIAL PROTEIN-SYNTHESIS IN RESPONSE TO SULFATE AND PHOSPHATE DEPRIVATION - IDENTIFICATION OF POSSIBLE COMPONENTS OF PLASMA-MEMBRANE TRANSPORT-SYSTEMS IN CULTURED TOMATO ROOTS

Isolated roots of Lycopersicon esculentum Mill., cultured in axenic conditions were starved of sulphate or phosphate, and uptake capacities for the respective oxyanion-transport systems were observed for several days after sulphate or phosphate withdrawal. Sulphate-uptake capacity of the intact roots, measured in a 20-min period, increased from a control level of 100 nmol.g-1.h-1 to 1100 nmol.g-1.h-1 in 10d, and phosphate-uptake capacity increased from 500 to 1400 nmol.g-1.h-1 over 4 d. Newly synthesised polypeptides of these root cultures were pulse-labelled in vivo for 2 h, by adding [H-3]leucine to the culture medium. The tissue was immediately homogenised and soluble and membrane fractions were prepared. A highly purified plasma-membrane fraction was separated from the crude microsomal membrane fraction using an aqueous two-phase partitioning technique. All fractions were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. A 28-kilodalton (kDa) soluble polypeptide, and 36-, 43-, and 47-kDa plasma-membrane polypeptides were observed to have increased labelling after 4 d of sulphate deprivation. Longer periods resulted in additional polypeptides with increased [H-3]leucine incorporation. The synthesis of a 25-kDa membrane polypeptide and a 65-kDa soluble polypeptide was increased after 4 d of phosphate deprivation. Two-dimensional electrophoresis afforded greater resolution of the plasma-membrane polypeptides, confirming increased synthesis of the 36-kDa polypeptide and the presence of the 28-kDa polypeptide in the plasma-membrane preparation from sulphate-starved roots. These polypeptides were also observed in protein-stained two-dimensional gels as low-abundant protein components of the plasma-membrane fraction. It is suggested that the 36-kDa polypeptide may be a component of the plasma-membrane sulphate-transport system and that the 25-kDa polypeptide may be a component of a phosphate-transport system.