Orthophosphate uptake in proteoid roots of naturally occurring Hakea sericea Schrad.

Abstract

P is an important macronutrient, making up about 0.2% of a plant’s dry weight. The form of P most readily accessed by plants is Pi, the concentration of which rarely exceeds 10 µM in soil (Schachtman et al., 1998). Here we studied the mechanisms involved on extraction of Pi from soil in Hakea sericea Schrad. (Proteacea), an Australian invader of natural habitats, which is able to develop proteoid roots as a response to P deficiency. Material and Methods: Proteoid roots were harvested from adult H. sericea shrubs growing in Serra d’Arga, Northern Portugal. The P uptake buffer contained 2.5-4000 µM NaH2PO4 and Pi concentration was determined according to Adams (1991). PCR amplification was performed using genomic DNA and degenerate primers based on the most conserved regions of plant P transporters genes. Results and Conclusions: H. sericea proteoid roots have highly efficient transporters for extraction of Pi from soil. Kinetic studies supported the involvement of two Pi mediated transport systems associated with a diffusion-like mechanism. The kinetic parameters were as follows: for the high-affinity system Km, 11 µM Pi and Vmax, 7 µmol h-1 g-1 FW; for the low-affinity system Km, 163 µM Pi and Vmax, 30 µmol h-1 g-1 FW. The protonophore CCCP (50 µM) inhibited up to 60% the initial uptake rates of 5-50 µM Pi (high-affinity range) and 100-500 µM (low-affinity range), suggesting H+-dependent transport. Both transporters were competitively inhibited by phosphite and 100 µM mersalyl reduced by 50% the initial uptake rates of 10 µM Pi. PCR using degenerate primers allowed the amplification of two 438 bp fragments sharing 87.4% identity with each other. Both nucleotide sequences comprise an incomplete ORF coding for transmembrane domains and exhibiting 85-82% identity with other plant P transporters. These nucleotide sequences are being used for the identification of complete cDNAs of Pi transporter genes of H. sericea.