Accumulation of salicylic acid and 4-hydroxybenzoic acid in phloem fluids of cucumber during systemic acquired resistance is preceded by a transient increase in phenylalanine ammonia-lyase activity in petioles and stems

Abstract

Cucumber (Cucumis sativa) leaves infiltrated with Pseudomonas syringae pv. syringae cells produced a mobile signal for systemic acquired resistance between 3 and 6 h after inoculation. The production of a mobile signal by inoculated leaves was followed by a transient increase in phenylalanine ammonia-lyase (PAL) activity in the petioles of inoculated leaves and in stems above inoculated leaves; with peaks in activity at 9 and 12 h, respectively, after inoculation. In contrast, PAL activity in inoculated leaves continued to rise slowly for at least 18 h. No increases in PAL activity were detected in healthy leaves of inoculated plants. Two benzoic acid derivatives, salicylic acid (SA) and 4-hydroxybenzoic acid (4HBA), began to accumulate in phloem fluids at about the time PAL activity began to increase, reaching maximum concentrations 15 h after inoculation. The accumulation of SA and 4HBA in phloem fluids was unaffected by the removal of all leaves 6 h after inoculation, and seedlings excised from roots prior to inoculation still accumulated high levels of SA and 4HBA. These results suggest that SA and 4HBA are synthesized de novo in stems and petioles in response to a mobile signal from the inoculated leaf.

Figure 1

Signal production by inoculated leaves. Cucumber seedlings were infiltrated on the second leaf with P. syringae cells at 50 sites per leaf. The inoculated leaves were removed from plants at various times and systemic chitinase accumulation was measured in the third leaf 5 d after inoculation. se values were smaller than the size of the symbols.

Figure 2

HPLC profile of phloem exudates. Phloem fluids were collected 18 h after infiltration of leaves with water (A and C) or with P. syringae (B and D). Eluting peaks were monitored for UV A₂₅₄ (A and B) and A₂₃₀ (C and D). The peaks were identified as SA glucoside (1), 4HBA (2), and SA (3).

Figure 3

Time course of SA and 4HBA accumulation in cucumber phloem exudates. The levels of SA (○, •) and 4HBA (□, ▪) in petiole phloem exudates from control (open symbols) or inoculated (closed symbols) plants were monitored for 36 h after infiltrating one leaf with water or with P. syringae cells. ses are indicated.

Figure 4

Accumulation of SA and 4HBA in phloem exudates after excision of leaves. Levels of SA and 4HBA in stem phloem exudate were determined 20 h after infiltration of one leaf on each plant with water (C) or P. syringae cells (I). Plants were either left intact (+Leaves), or had all leaves excised 6 h after inoculation (−Leaves). ses are indicated.

Figure 5

Effect of excision of seedlings from roots on the accumulation of SA and 4HBA. Seedlings were either left intact (+Roots) or were excised from roots at the stem base and placed in water 2 d prior to inoculation (−Roots). Levels of SA and 4HBA in stem phloem exudate were determined 20 h after infiltration of one leaf on each plant with water (C) or with P. syringae (I). ses are indicated.

Figure 6

Time course of PAL activity in different tissues during SAR. PAL activity was measured in leaves infiltrated with P. syringae (•) or water (□) (Inoculated Leaf); in 1-cm-long petiole segments of inoculated leaves (Petiole of Inoculated Leaf); or in 1-cm-long stem segments immediately above inoculated leaves (Stem) at various times after inoculation. ses are indicated.

Figure 7

Effect of leaf excision on petiole PAL activity. Seedlings inoculated with P. syringae or water were either left intact (+Leaf) or the inoculated leaves were excised at the leaf base 6 h after inoculation (−Leaf). PAL activity was measured in 1-cm segments taken from the base of the petiole adjacent to the stem at 6 and 9 h after inoculation. ses are indicated.

Figure 8

Proposed pathways of SA and 4HBA biosynthesis. Enzymatic steps for which the enzymes have been identified include PAL, CA4H (cinnamic acid 4-hydroxylase), and BA2H (benzoic acid 2-hydroxylase).