Rapid alkalinization factors in poplar cell cultures. Peptide isolation,cDNA cloning,and differential expression in leaves and methyl jasmonate-treated cells

A family of peptides inducing rapid pH alkalinization in hybrid poplar (Populus trichocarpa x Populus deltoides) cell culture medium was isolated from hybrid poplar leaves. Five related approximately 5-kD peptides were purified by high-performance liquid chromatography and analyzed by matrix-assisted laser desorption ionization-mass spectrometry. The N-terminal sequence of one of the isolated peptides was very similar to a previously characterized peptide from tobacco (Nicotiana tabacum), rapid alkalinization factor (RALF), which causes a rapid increase in culture medium pH when added to tobacco cell cultures (G. Pearce, D.S. Moura, J. Stratmann, C.A. Ryan [2001] Proc Natl Acad Sci USA 98: 12843-12847). Two unique poplar RALF cDNAs (PtdRALF1 and PtdRALF2) were isolated from a poplar cDNA library and used to study RALF expression in poplar saplings and cultured poplar cells. Both genes were found to be expressed constitutively in poplar saplings and cultured cells. However, PtdRALF2 was expressed in leaves at very low levels, and its expression in suspension culture cells was transiently suppressed by methyl jasmonate (MeJa). Although the function of these novel peptides remains enigmatic, our experiments suggest their role may be developmental rather than stress related. Overall, our study confirms the presence of active RALF peptides in other plants, and provides new data on the complexity of the RALF gene family in poplar.