The production of ROS and NO during stomatal closure by variable signals

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Effect of different modulators on 10 µm abscisic acid (ABA)-induced nitric oxide (NO) production, as indicated by 4,5-diaminofluorescein diacetate (DAF-2DA) fluorescence in stomatal guard cells of Pisum sativum. (a) to (g) are the controls: treated with water (a), 0.1 mmsodium nitroprusside (SNP) (b), 0.1 mm butyrate (c), 2 mm methylamine (d), 0.2 mm cPTIO (e), 0.1 mm L-NAME (f) and 1 mm EGTA (g) in the absence of ABA, respectively. (h) to (n) are epidermal strips treated with ABA, as follows: ABA alone (h), ABA along with 0.1 mmSNP (i), 0.1 mm butyrate (j), 2 mm methylamine (k), 0.2 mm cPTIO (l), 0.1 mm L-NAME (m) and 1 mm EGTA (n) in the presence of ABA, respectively. Confocal fluorescence images were taken at 18 min after addition of 10 µm ABA. Further details are given in the Materials and Methods section. Bar = 25 µm.

Cytosolic alkalinization is a common and early messenger preceding the production of ROS and NO during stomatal closure by variable signals, including abscisic acid, methyl jasmonate and chitosan

by Gonugunta V. K., Srivastava N., Raghavendra A. S. (2009)

in Plant Signaling & Behavior 4, 561–564.

| CrossRef | CAS |

Confocal fluorescence images of stomata stained with 2′,7′-bis(2-carboxy-ethyl)-5(6)-carboxy fluorescein-acetoxy methyl ester (BCECF-AM) (a,c) or 4,5-diaminofluorescein diacetate (DAF-2DA) (b,d). These were taken after 12 min for BCECF-AM and 18 min for DAF-2DA treatment with 10 µm abscisic acid (ABA). (a) and (b) are the controls, while (c) and (d) are the stomata treated with ABA. Bar = 25 µm. –
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Abstract

Abscisic acid (ABA) raised the cytosolic pH and nitric oxide (NO) levels in guard cells while inducing stomatal closure in epidermis of Pisum sativum.

Butyrate (a weak acid) reduced the cytosolic pH/NO production and prevented stomatal closure by ABA. Methylamine (a weak base) enhanced the cytosolic alkalinization and aggravated stomatal closure by ABA. The rise in guard cell pH because of ABA became noticeable after 6 min and peaked at 12 min, while NO production started at 9 min and peaked at 18 min.

These results suggested that NO production was downstream of the rise in cytosolic pH. The ABA-induced increase in NO of guard cells and stomatal closure was prevented by 2-phenyl-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide (cPTIO, a NO scavenger) and partially by N-nitro-L-Arg-methyl ester (L-NAME, an inhibitor of NO synthase). In contrast, cPTIO or L-NAME had only a marginal effect on the pH rise induced by ABA. Ethylene glycol tetraacetic acid (EGTA, a calcium chelator) prevented ABA-induced stomatal closure while restricting cytosolic pH rise and NO production.

We suggest that during ABA-induced stomatal closure, a rise in cytosolic pH is necessary for NO production. Calcium may act upstream of cytosolic alkalinization and NO production, besides its known function as a downstream component.

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