RALF, a 5-kDa ubiquitous polypeptide in plants, arrests root growth and development

Abstract

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.

Figure 1

Purification of RALF by HPLC. Activity was determined by adding 2–10 μl of each fraction from HPLC separations to 1 ml of tobacco cells and measuring the pH of the media after 15 min (Lower). (A) Semipreparative C18 HPLC purification. Fractions 77–78 were pooled and lyophilized. (B) Strong cation exchange HPLC purification. Fractions 50–51 were pooled and lyophilized. (C) Narrow-bore C18 HPLC purification. Fractions 61–63 were pooled and lyophilized.

Figure 2

Biological activities of the 5-kDa polypeptide from tobacco leaves. (A) A comparison of the alkalinization of tobacco suspension-cultured cell medium by the synthetic 5-kDa alkalinating factor with two tobacco systemins (2). Each polypeptide was assayed at a concentration of 1 nM. (B) MAP kinase activity in tobacco suspension-cultured cells treated with the 5-kDa polypeptide (AF) compared with the activity induced by two tobacco systemins. [Reproduced with permission from ref. (Copyright 2001, Macmillan Magazines Ltd.).]

Figure 3

The nucleotide and deduced protein sequences of RALF precursor cDNA. The RALF polypeptide sequence is at the C terminus of the preproprotein and is underlined. Arrow indicates the predicted signal peptide cleavage site.

Figure 4

The amino acid sequence of tobacco RALF compared with known EST sequences.

Figure 5

Comparison of the alkalinization response of tomato suspension cells with native, synthetic, and alkylated RALF. The pH responses of the synthetic peptides were compared with native tomato RALF after 10 min.

Figure 6

Effects of water, 10 μM RALF, and 10 μM alkRALF on germinating seedlings. Seeds were germinated in water for 2 days and then treated as above for 2 days. (A) Tomato seedlings. A severe arrest of root growth and development can be seen in tomato seedlings treated with RALF. (B) Arabidopsis seedlings treated as in A. Photos are of Arabidopsis seedlings taken at different magnifications.