Differential Metabolisms of Green Leaf Volatiles in Injured and Intact Parts of a Wounded Leaf Meet Distinct Ecophysiological Requirements

Almost all terrestrial plants produce green leaf volatiles (GLVs), consisting of six-carbon (C6) aldehydes, alcohols and their esters, after mechanical wounding. C6 aldehydes deter enemies, but C6 alcohols and esters are rather inert. In this study, we address why the ability to produce various GLVs in wounded plant tissues has been conserved in the plant kingdom. The major product in completely disrupted Arabidopsis leaf tissues was (Z)-3-hexenal, while (Z)-3-hexenol and (Z)-3-hexenyl acetate were the main products formed in the intact parts of partially wounded leaves. 13C-labeled C6 aldehydes placed on the disrupted part of a wounded leaf diffused into neighboring intact tissues and were reduced to C6 alcohols. The reduction of the aldehydes to alcohols was catalyzed by an NADPH-dependent reductase. When NADPH was supplemented to disrupted tissues, C6 aldehydes were reduced to C6 alcohols, indicating that C6 aldehydes accumulated because of insufficient NADPH. When the leaves were exposed to higher doses of C6 aldehydes, however, a substantial fraction of C6 aldehydes persisted in the leaves and damaged them, indicating potential toxicity of C6 aldehydes to the leaf cells. Thus, the production of C6 aldehydes and their differential metabolisms in wounded leaves has dual benefits. In disrupted tissues, C6 aldehydes and their α,β-unsaturated aldehyde derivatives accumulate to deter invaders. In intact cells, the aldehydes are reduced to minimize self-toxicity and allow healthy cells to survive. The metabolism of GLVs is thus efficiently designed to meet ecophysiological requirements of the microenvironments within a wounded leaf

Changes in photosystem II activity in <i>Arabidopsis</i> leaves during exposure to C6-volatiles.

<p><b>A: </b><i>Arabidopsis</i> (<i>Col-0</i>) plants were exposed to vapor of CH₂Cl₂ (control, triangles), (<i>Z</i>)-3-hexenol (open circles), (<i>E</i>)-2-hexenal (squares), or (<i>Z</i>)-3-hexenal (closed circles) in the light (60 µmol m⁻² s⁻¹, supplied by white fluorescent lamps). PSII activity was determined using the chlorophyll fluorescence parameter <i>F_V/F_M</i>, detected with a MINI-PAM Chlorophyll Fluorometer. Plants were incubated in darkness for 5 min before each measurement. Error bars represent SE (<i>n</i> = 12). Data were analyzed by Tukey's test. Different letters indicate significant differences among treatments at different times (<i>P</i><0.05). <b>B:</b> Representative plants after 22 h of exposure to the vapors. Note the yellowing and wilding of leaves exposed to (<i>E</i>)-2-hexenal and (<i>Z</i>)-3-hexenal. <b>C:</b> Plants were exposed to different concentrations of CH₂Cl₂ (control, withe bars) or (<i>Z</i>)-3-hexenal (shaded bars) for 10 min before <i>F_V/F_M</i> was measured. Significant differences between the control and the treated plants are indicated by asterisks (*, <i>P</i><0.05; **, <i>P</i><0.01; <i>t</i>-test).</p

Diffusion of ¹³C-<i>n</i>-hexanal and (<i>Z</i>)-3-hexenal into neighboring intact tissues.

a<p>Isotope enrichment of each compound was more than 95%.</p>b<p>Values are mean ± SE (n = 3).</p

Conversion of (<i>Z</i>)-3-hexenal in intact <i>Arabidopsis</i> (<i>Col-0</i>).

<p>Intact <i>Arabidopsis</i> (<i>Col-0</i>) plants were exposed to (<i>Z</i>)-3-hexenal vapor (1.0 nmol cm⁻³), transferred into a fresh glass container, and the amounts of (<i>Z</i>)-3-hexenyl acetate (white bars) and (<i>Z</i>)-3-hexenol (black bars) emitted from the plants were quantified. Control plants were exposed to CH₂Cl₂ vapor (carrier solvent for volatiles). Error bars represent SE (<i>n</i> = 3). Data were analyzed by Tukey's test. Different letters above the bars indicate significant differences among treatments in the emission of each compound (<i>P</i><0.05).</p

Biochemical pathway for the formation of (<i>Z</i>)-3-hexenal and its related compounds.

<p>In the biochemical pathway for formation of (<i>Z</i>)-3-hexenal and its related compounds, lipoxygenase adds dioxygen at position 13 of linolenic acid to produce linolenic acid 13-hydroperoxide. The hydroperoxide is cleaved by hydroperoxide lyase at the C12–C13 bond to produce (<i>Z</i>)-3-hexenal, which can be reduced to form (<i>Z</i>)-3-hexenol. A portion of (<i>Z</i>)-3-hexenol is further converted to (<i>Z</i>)-3-hexenyl acetate. In some plants, (<i>Z</i>)-3-hexenal is converted to (<i>E</i>)-2-hexenal spontaneously or enzymatically. (Z)-3-Hexenal is spontaneously oxygenated to form 4-hydroperoxy-(<i>E</i>)-2-hexenal, 4-hydroxy-(<i>E</i>)-2-hexenal or 4-oxo-(<i>E</i>)-2-hexenal.</p

Conversion of U-¹³C-<i>n</i>-hexanal and U-¹³C-(<i>Z</i>)-3-hexenal over time by intact <i>Arabidopsis</i> plants.

<p>An intact <i>Arabidopsis</i> (<i>Col-0</i>) plant or a pot with soil (control) was placed in a plastic box with a cotton swab containing 2 µl of a mixture containing U-¹³C-<i>n</i>-hexanal and (<i>Z</i>)-3-hexenal. At 0, 10, or 30 min after the onset of exposure, an SPME fiber was positioned in the headspace of the box and volatiles were collected for 10 min. <b>A:</b> Typical chromatograms obtained after 40 min exposure. Compound numbers are as in the Fig. 2 legend. Numbers with prime marks indicate the corresponding ¹³C-labeled compound (>95% enrichment). The amounts of volatile aldehydes (<b>B</b>), volatile alcohols (<b>C</b>), and volatile acetates (<b>D</b>) were quantified from calibration curves constructed with authentic compounds. Error bars represent SE (<i>n</i> = 3). Data were analyzed by Tukey's test. Different letters above the bars indicate significant differences among the sampling times for each compound (<i>P</i><0.05). nd: not detected.</p

Conversion of volatile aldehydes to their corresponding alcohols in the presence of NADPH or NADH.

<p><i>Arabidopsis</i> (<i>No-0</i>) leaves were disrupted completely in the absence (control, white bars) or presence of NADH (+NADH, shaded bars) or NADPH (+NADPH, black bars), and the volatile compounds that formed were analyzed. Inset: amounts of some volatile compounds quantified from corresponding calibration curves. Error bars represent SE (<i>n</i> = 3). Asterisks indicate significant differences compared with the control (<i>P</i><0.05, <i>t</i>-test).</p

Localization of volatile compounds after exposure to (<i>Z</i>)-3-hexenal vapor.

<p>Intact leaves exposed to (<i>Z</i>)-3-hexenal vapor (0, 0.01, 0.1, and 1.0 mmol cm⁻³) were washed for 10 s with methanol to extract methanol-soluble surface volatiles (leaf wash) and the remaining surface-washed leaves were extracted with methyl <i>tert</i>-butyl ether (leaf extract). Error bars represent SE (<i>n</i> = 3). Data were analyzed by Tukey's test. Letters indicate significant differences in amounts of extracted compounds among exposure concentrations (<i>P</i><0.05).</p