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Phospholipase A2beta mediates light-induced stomatal opening in Arabidopsis.

Seo J, Lee HY, Choi H, Choi Y, Lee Y, Kim YW, Ryu SB, Lee Y - J. Exp. Bot. (2008)

Bottom Line: To identify PLA(2) gene(s) that is (are) involved in light-induced stomatal opening, stomatal movement was examined in Arabidopsis thaliana plants in which the expression of PLA(2) isoforms was reduced or knocked-out.Aristolochic acid, which inhibits light-induced stomatal opening, inhibited the activity of purified PLA(2)beta.Collectively, these results provide evidence that PLA(2)beta is involved in light-induced stomatal opening in Arabidopsis.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular and Life Sciences, POSTECH, Pohang 790-784, Korea.

ABSTRACT
Phospholipase A(2) (PLA(2)) catalyses the hydrolysis of phospholipids into lysophospholipids and free fatty acids. Physiological studies have indicated that PLA(2) is involved in stomatal movement. However, genetic evidence of a role of PLA(2) in guard cell signalling has not yet been reported. To identify PLA(2) gene(s) that is (are) involved in light-induced stomatal opening, stomatal movement was examined in Arabidopsis thaliana plants in which the expression of PLA(2) isoforms was reduced or knocked-out. Light-induced stomatal opening in PLA(2)alpha knockout plants did not differ from wild-type plants. Plants in which PLA(2)beta was silenced by RNA interference exhibited delayed light-induced stomatal opening, and this phenotype was reversed by exogenous lysophospholipids, which are products of PLA(2). Stomatal opening in transgenic plants that over-expressed PLA(2)beta was faster than wild-type plants. The expression of PLA(2)beta was localized to the endoplasmic reticulum of guard cells, and increased in response to light in the mature leaf. Aristolochic acid, which inhibits light-induced stomatal opening, inhibited the activity of purified PLA(2)beta. Collectively, these results provide evidence that PLA(2)beta is involved in light-induced stomatal opening in Arabidopsis.

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The effect of aristolochic acid (Aris), a low molecular weight PLA2 inhibitor, on the activity of purified PLA2β. (A) Light-induced stomatal opening of wild-type Arabidopsis in the presence or absence of 20 μM Aris. (B) Thin layer chromatography (TLC) analysis of the hydrolytic activity of recombinant PLA2β. Purified PLA2β was incubated in 100 mM TRIS–HCl (pH 8.0) at 40 °C in the presence or absence of 20 μM Aris for 40 min, and then PLA2β hydrolytic activity was assayed in the presence of 1-palmitoyl-2-[14C]palmitoyl-PC. The acyl-hydrolysis activity of the recombinant protein was greatly inhibited by Aris, compared to the solvent controls. Lane 1, substrate only; lanes 2–3, solvent controls (40 μM NaOH); lanes 4–5, 20 μM Aris dissolved in NaOH; lane 6, bee venom low molecular weight secretory PLA2 was used instead of purified PLA2β to identify the position of 14C-palmitic acid in the TLC plate. (C) The results from TLC were quantified using a phosphoimager (n=4).
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fig4: The effect of aristolochic acid (Aris), a low molecular weight PLA2 inhibitor, on the activity of purified PLA2β. (A) Light-induced stomatal opening of wild-type Arabidopsis in the presence or absence of 20 μM Aris. (B) Thin layer chromatography (TLC) analysis of the hydrolytic activity of recombinant PLA2β. Purified PLA2β was incubated in 100 mM TRIS–HCl (pH 8.0) at 40 °C in the presence or absence of 20 μM Aris for 40 min, and then PLA2β hydrolytic activity was assayed in the presence of 1-palmitoyl-2-[14C]palmitoyl-PC. The acyl-hydrolysis activity of the recombinant protein was greatly inhibited by Aris, compared to the solvent controls. Lane 1, substrate only; lanes 2–3, solvent controls (40 μM NaOH); lanes 4–5, 20 μM Aris dissolved in NaOH; lane 6, bee venom low molecular weight secretory PLA2 was used instead of purified PLA2β to identify the position of 14C-palmitic acid in the TLC plate. (C) The results from TLC were quantified using a phosphoimager (n=4).

Mentions: Aristolochic acid (Aris) has previously been shown to inhibit light-induced stomatal opening in Commelina communis (Suh et al., 1998). It was also tested whether or not Aris inhibits stomatal opening in Arabidopsis. The stomata of Aris-treated Arabidopsis guard cells opened more slowly than the stomata of untreated cells in response to light (Fig. 4A). To determine whether the activity of PLA2β is affected by this inhibitor, the effect of Aris on the activity of purified PLA2β was examined using a radiolabelled substrate, 1-palmitoyl-2-[14C]palmitoyl-PC. In control samples, the radioactive substrate was hydrolysed into the corresponding FFA, and readily detectable by thin-layer chromatography (TLC) (Fig. 4B, lanes 2 and 3). The amount of radiolabelled FFA decreased (Fig. 4B, lanes 4 and 5) to 38±1.8% (average ±SE, Fig. 4C) when PLA2β was preincubated with Aris, which indicated that PLA2β activity was strongly inhibited. These results suggested that the inhibitory effect of Aris on light-induced stomatal opening is due to its ability to inhibit PLA2β.


Phospholipase A2beta mediates light-induced stomatal opening in Arabidopsis.

Seo J, Lee HY, Choi H, Choi Y, Lee Y, Kim YW, Ryu SB, Lee Y - J. Exp. Bot. (2008)

The effect of aristolochic acid (Aris), a low molecular weight PLA2 inhibitor, on the activity of purified PLA2β. (A) Light-induced stomatal opening of wild-type Arabidopsis in the presence or absence of 20 μM Aris. (B) Thin layer chromatography (TLC) analysis of the hydrolytic activity of recombinant PLA2β. Purified PLA2β was incubated in 100 mM TRIS–HCl (pH 8.0) at 40 °C in the presence or absence of 20 μM Aris for 40 min, and then PLA2β hydrolytic activity was assayed in the presence of 1-palmitoyl-2-[14C]palmitoyl-PC. The acyl-hydrolysis activity of the recombinant protein was greatly inhibited by Aris, compared to the solvent controls. Lane 1, substrate only; lanes 2–3, solvent controls (40 μM NaOH); lanes 4–5, 20 μM Aris dissolved in NaOH; lane 6, bee venom low molecular weight secretory PLA2 was used instead of purified PLA2β to identify the position of 14C-palmitic acid in the TLC plate. (C) The results from TLC were quantified using a phosphoimager (n=4).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2561155&req=5

fig4: The effect of aristolochic acid (Aris), a low molecular weight PLA2 inhibitor, on the activity of purified PLA2β. (A) Light-induced stomatal opening of wild-type Arabidopsis in the presence or absence of 20 μM Aris. (B) Thin layer chromatography (TLC) analysis of the hydrolytic activity of recombinant PLA2β. Purified PLA2β was incubated in 100 mM TRIS–HCl (pH 8.0) at 40 °C in the presence or absence of 20 μM Aris for 40 min, and then PLA2β hydrolytic activity was assayed in the presence of 1-palmitoyl-2-[14C]palmitoyl-PC. The acyl-hydrolysis activity of the recombinant protein was greatly inhibited by Aris, compared to the solvent controls. Lane 1, substrate only; lanes 2–3, solvent controls (40 μM NaOH); lanes 4–5, 20 μM Aris dissolved in NaOH; lane 6, bee venom low molecular weight secretory PLA2 was used instead of purified PLA2β to identify the position of 14C-palmitic acid in the TLC plate. (C) The results from TLC were quantified using a phosphoimager (n=4).
Mentions: Aristolochic acid (Aris) has previously been shown to inhibit light-induced stomatal opening in Commelina communis (Suh et al., 1998). It was also tested whether or not Aris inhibits stomatal opening in Arabidopsis. The stomata of Aris-treated Arabidopsis guard cells opened more slowly than the stomata of untreated cells in response to light (Fig. 4A). To determine whether the activity of PLA2β is affected by this inhibitor, the effect of Aris on the activity of purified PLA2β was examined using a radiolabelled substrate, 1-palmitoyl-2-[14C]palmitoyl-PC. In control samples, the radioactive substrate was hydrolysed into the corresponding FFA, and readily detectable by thin-layer chromatography (TLC) (Fig. 4B, lanes 2 and 3). The amount of radiolabelled FFA decreased (Fig. 4B, lanes 4 and 5) to 38±1.8% (average ±SE, Fig. 4C) when PLA2β was preincubated with Aris, which indicated that PLA2β activity was strongly inhibited. These results suggested that the inhibitory effect of Aris on light-induced stomatal opening is due to its ability to inhibit PLA2β.

Bottom Line: To identify PLA(2) gene(s) that is (are) involved in light-induced stomatal opening, stomatal movement was examined in Arabidopsis thaliana plants in which the expression of PLA(2) isoforms was reduced or knocked-out.Aristolochic acid, which inhibits light-induced stomatal opening, inhibited the activity of purified PLA(2)beta.Collectively, these results provide evidence that PLA(2)beta is involved in light-induced stomatal opening in Arabidopsis.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular and Life Sciences, POSTECH, Pohang 790-784, Korea.

ABSTRACT
Phospholipase A(2) (PLA(2)) catalyses the hydrolysis of phospholipids into lysophospholipids and free fatty acids. Physiological studies have indicated that PLA(2) is involved in stomatal movement. However, genetic evidence of a role of PLA(2) in guard cell signalling has not yet been reported. To identify PLA(2) gene(s) that is (are) involved in light-induced stomatal opening, stomatal movement was examined in Arabidopsis thaliana plants in which the expression of PLA(2) isoforms was reduced or knocked-out. Light-induced stomatal opening in PLA(2)alpha knockout plants did not differ from wild-type plants. Plants in which PLA(2)beta was silenced by RNA interference exhibited delayed light-induced stomatal opening, and this phenotype was reversed by exogenous lysophospholipids, which are products of PLA(2). Stomatal opening in transgenic plants that over-expressed PLA(2)beta was faster than wild-type plants. The expression of PLA(2)beta was localized to the endoplasmic reticulum of guard cells, and increased in response to light in the mature leaf. Aristolochic acid, which inhibits light-induced stomatal opening, inhibited the activity of purified PLA(2)beta. Collectively, these results provide evidence that PLA(2)beta is involved in light-induced stomatal opening in Arabidopsis.

Show MeSH
Related in: MedlinePlus