A 5-kDa polypeptide was isolated from tobacco leaves that induced a rapid alkalinization of the culture medium of tobacco suspension-cultured cells and a concomitant activation of an intracellular mitogen-activated protein kinase. An N-terminal sequence was obtained, and a cDNA coding for the 49-aa polypeptide was isolated from a tobacco cDNA library. The cDNA encoded a preproprotein of 115 amino acids that contained the polypeptide at its C terminus. A search among known expressed sequence tags revealed that genes encoding Rapid ALkalinization Factor (RALF) preproproteins were present in various tissues and organs from 16 species of plants representing 9 families. A tomato homolog of the polypeptide was synthesized and, when supplied to germinating tomato and Arabidopsis seeds, it caused an arrest of root growth and development. Although its specific role in growth has not been established, the polypeptide joins the ranks of the increasing number of polypeptide hormones that are known to regulate plant stress, growth, and development.

A photoaffinity analog of tomato leaf RALF peptide (LeRALF), (125)I-azido-LeRALF, bound saturably to tomato suspension cultured cells in the dark in a classical receptor binding assay. Classical kinetic analyses revealed that the analog interacted with a single binding site on the surface of the cells with a KD of 0.8x10(-9) M, typical of known peptide hormone-receptor interactions in both plants and animals. The (125)I-azido-LeRALF, when exposed to UVB light in the presence of the cells, strongly labeled only two proteins of 25 kDa and 120 kDa, with the 25 kDa protein being more strongly labeled than the 120 kDa protein. The cell-surface localization of the interaction was indicated, as suramin, a known inhibitor of peptide-receptor interactions, and native LeRALF peptide competed with (125)I-azido-LeRALF labeling of both proteins. Two biologically inactive LeRALF analogs were not competitors. Incubation of (125)I-azido-LeRALF with suspension cultured cells in the dark, where it was fully active, could subsequently be totally dissociated from cells by acid washes, indicating that it was interacting at the cell surface and was not internalized. The (125)I-azido-LeRALF-labeled 25 kDa and 120 kDa proteins could not be solubilized from cell membranes by methods that release peripheral proteins, indicating that they are integral membrane components. The cumulative kinetic and biochemical evidence strongly indicates that the two proteins may be components of a LeRALF receptor complex.

Rapid Alkalinization Factors (RALFs) are plant peptides that rapidly increase the pH of plant suspension cell culture medium and inhibit root growth. A pollen-specific tomato (Solanum lycopersicum) RALF (SlPRALF) has been identified. The SlPRALF gene encodes a preproprotein that appears to be processed and released from the pollen tube as an active peptide. A synthetic SlPRALF peptide based on the putative active peptide did not affect pollen hydration or viability, but inhibited the elongation of normal pollen tubes in an in vitro growth system. Inhibitory effects of SlPRALF were detectable at concentrations as low as 10 nM and complete inhibition was observed at 1 muM peptide. At least ten-fold higher levels of alkSlPRALF, which lacks disulfide bonds, were required to see similar effects. A greater effect of peptide was observed in low pH buffered medium. Inhibition of pollen tube elongation was reversible if peptide was removed within 15 min of exposure. Addition of 100 nM SlPRALF to actively growing pollen tubes inhibited further elongation until tubes were 40 to 60 mum in length, after which pollen tubes became resistant to the peptide. The onset of resistance correlated with the timing of the exit of the Male Germ Unit (MGU) from the pollen grain into the tube. Thus, exogenous SlPRALF acts as a negative regulator of pollen tube elongation within a specific developmental window.