Myelin basic protein kinase activity in tomato leaves is induced systemically by wounding and increases in response to systemin and oligosaccharide elicitors

Abstract

In response to wounding, a 48-kDa myelin basic protein (MBP) kinase is activated within 2 min, both locally and systemically, in leaves of young tomato plants. The activating signal is able to pass through a steam girdle on the stem, indicating that it moves through the xylem and does not require intact phloem tissue. A 48-kDa MBP kinase is also activated by the 18-amino acid polypeptide systemin, a potent wound signal for the synthesis of systemic wound response proteins (swrps). The kinase activation by systemin is strongly inhibited by a systemin analog having a Thr-17 --> Ala-17 substitution, which is a powerful antagonist of systemin activation of swrp genes. A 48-kDa MBP kinase activity also increases in response to polygalacturonic acid and chitosan but not in response to jasmonic acid or phytodienoic acid. In def1, a mutant tomato line having a defective octadecanoid pathway, the 48-kDa MBP kinase is activated by wounding and systemin as in the wild-type plants. This indicates that MBP kinase functions between the perception of primary signals and the DEF1 gene product. In response to wounding, the MBP kinase is phosphorylated on phosphotyrosine residues, indicating a relationship to the mitogen-activated protein kinase family of protein kinases.

Figure 1

A 48-kDa MBPK is systemically activated by wounding in both the wounded and in unwounded leaves. The lower leaf of 14- to 15-day-old tomato plants (two expanded leaves) was wounded with a hemostat and incubated under standard conditions as described. At the times indicated, the lower wounded and upper unwounded leaves were excised at the base of the petiole and assayed in an in-gel MBPK assay (A) and quantified using an InstantImager (B) with kinase activities expressed as the fold activity above the level of untreated control leaves, which were assigned a value of 1. ▪, Wounded leaves; •, systemic unwounded leaves.

Figure 2

A 48-kDa MBPK is activated by systemin. Young (14 days old) tomato plants were excised at the base of the stem and the cut end placed into a solution containing either 25 nM systemin in 10 mM phosphate buffer (pH 6.5) or buffer alone and incubated under standard conditions. At the times indicated, the leaves were assayed as in Fig. 1A for MBPK activity.

Figure 3

Comparison of the induction of MBPK activity and of inhibitor I protein by systemin in leaves of excised 14-day-old tomato plants. Plants were supplied with systemin in 10 mM phosphate buffer (pH 6.5) or buffer alone. At 30 min, leaves were assayed for MBPK activity (○) and at 24 hr leave juice was assayed for inhibitor I protein accumulation (•). The kinase activities are expressed as the fold activation above or below the level of the buffer control, which was assigned a value of 1.

Figure 4

Inhibition of the induction of MBPK activity and of inhibitor I protein by the systemin analog, Ala-17-systemin. Young excised tomato plants were supplied with 10 mM phosphate buffer, pH 6.5 (B); 25 nM systemin in buffer (SYS); 2.5 μM Ala-17-systemin in buffer (A17); and 25 nM systemin plus 2.5 μM Ala-17-systemin in buffer (A17 + SYS). The plants were incubated under standard conditions and assayed for MBPK activity (solid bars) after 30 min and for inhibitor I protein accumulation after 24 hr (shaded bars). The kinase activities are expressed as the fold activation above or below the level of the buffer control, which was assigned a value of 1.

Figure 5

The 48-kDa MBPK is activated by wounding and systemin in the octadecanoid signaling mutant def1. Mutant plants (14 days old) were wounded on all leaves with a hemostat across the main veins. A second set of plants were excised at the base of the stem and supplied with a 25 nM systemin solution in 10 mM phosphate buffer (pH 6.5). All plants were incubated under standard conditions, and at the times indicated the leaves were assayed for MBPK activity.

Figure 6

A wound signal and systemin both pass a steam girdle on the stem. Young plants (14 days old) were girdled with a steam jet as described to produce a 0.5–1.0 cm zone comprised only of a thin bundle of intact xylem vessels. On the following day, the girdled plants were excised either below or above the steam girdle and placed with their cut stems into a solution containing either 10 mM phosphate buffer (pH 6.5) or buffer containing 25 nM systemin and incubated under standard conditions. At the times indicated, leaves were assayed for MBPK activity.

Figure 7

MBPK is systemically activated by M. sexta larvae attack. A single M. sexta larva (≈7 cm long) was placed near the exposed edge of a terminal leaflet of the lower leaf and allowed to chew continuously for 3 min without other contact with the leaf (see Materials and Methods). The wounded leaf (lane W), the upper unwounded leaf (lane U), and untreated control leaves (lane C) were assayed for MBPK activity.

Figure 8

The wound-activated MBPK is phosphorylated on tyrosine residues. Extracts were prepared from wounded 14-day-old tomato plants 10 min following wounding (lanes W) and from unwounded control plants (lanes C) as described. The proteins were immunoprecipitated with a monoclonal antibody specific for phosphotyrosine (mAb anti-phosphotyrosine), and the precipitated proteins were assayed for MBPK activity. The amino acids phosphotyrosine or phosphothreonine were added as competitors to demonstrate the specificity of the antibody.