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

Abstract

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.

Figure 1

SCX HPLC of RALFs from poplar leaf extracts. Active compounds were separated using a 350 to 650 mm KCl gradient (dashed line). Medium pH alkalinization activity was assayed by adding 2 μL of each fraction to 2 mL of cells and measuring the pH every 5 min for 35 min. The maximum pH increase (ΔpH) was calculated and plotted with the elution profile monitored at 215 nm. HPLC peaks 1 through 3 (small solid peaks in the elution profile) that corresponded to the highest alkalinization activity were recovered and further purified by C18 HPLC.

Figure 2

Medium alkalinization in poplar suspension culture in response to RALF and elicitors. Cell culture medium was monitored every 5 min. RALF1, flg22, and pep13 peptides were assayed at concentrations of 1 nm, 1 μm, and 5 μm, respectively. Chitosan was assayed at a final concentration of 1 μg mL⁻¹.

Figure 3

Deduced amino acid sequences of two PtdRALF cDNAs compared with the aspen EST sequence (accession no. AI163551) and the tobacco RALF sequence (accession no. AF407278). The N-terminal sequence determined by Edman sequencing of RALF1 is underlined. The triangles indicate conserved Cys residues. Regions underscored with a dashed line correspond to sequences used for PCR primers. The GenBank accession numbers are AY172330 and AY172331 for PtdRALF1 and PtdRALF2, respectively.

Figure 4

Southern analysis of the RALF gene family in poplar. Ten micrograms of restricted genomic DNA was probed with the full-length PtdRALF1 cDNA (left). The same membrane was stripped and rehybridized with the PtdRALF2 cDNA (right). U, Undigested; H, HindIII; X, XbaI; E, EcoRV.

Figure 5

Northern analysis of PtdRALF expression. RNA was extracted from tissues of different ages from a 3-month-old sapling. Total RNA (20 μg lane⁻¹) was blotted onto membranes and hybridized with gene-specific probes for PtdRALF1 or PtdRALF2 (see “Materials and Methods”). EtBr, Ethidium bromide-stained gel; Ap, apical tissue; Pe, petiole; Le, leaf; St, stem; Ba, bark; Wo, wood; Rt, root tip, Ro, root; Bu, bud.

Figure 6

Northern analysis of PtdRALF in poplar cells after various treatments. Three-day-old cultures were treated for 5 h with compounds at concentrations as described in “Materials and Methods,” and analyzed for PtdRALF1 and PtdRALF2 gene expression. EtBr, Ethidium bromide-stained gel; Co, control; BA, benzyl adenine; NA, naphthalene acetic acid; MJ, MeJa; Suc, Suc; N, ammonium nitrate; Ch, chitosan; Pm, P. megasperma elicitor; HCl; hydrochloric acid.

Figure 7

Northern analysis showing the effect of MeJa and PtdRALF2 expression in poplar cell cultures. Cultures (40 mL) were treated with 50 μm MeJa, and at the times indicated the cells were harvested for RNA extraction and the medium pH was measured. The ethidium-stained gel is shown on the lower panel as a loading control.