Effects of chemical inhibitors and apyrase enzyme further document a role for apyrases and extracellular ATP in the opening and closing of stomates inArabidopsis

Abstract

Extracellular ATP (eATP), which has now become recognized as a signaling agent in plants, can regulate growth in a variety of plant cell and tissue types. 1 The application of micromolar concentrations of ATP can induce [Ca 2+ ] cyt fluctuations and promote growth-altering accumulation of ROS and NO in diverse tissues of diverse plants. Nitric oxide (NO) and reactive oxygen species (ROS) are important in guard cell responses. In our original study of this signaling response we found that ATPγS-induced stomatal closing was dependent on the production of ROS and NO.2 Importantly we reported that both ABA and light-induced changes in stomatal aperture were preceded by an increase in the eATP levels of guard cells. A subsequent report by Hao et al. (2012) 3 found that applied ATP promoted stomatal opening, but, in contrast to the results we obtained using ATPγS, they did not observe stomatal closing in Arabidopsis or Vicia faba leaves in response to applied ATP. In this study we carry out additional tests that address questions raised by the findings of Hao et al. Regarding eATP-induced stomatal opening, we hypothesized that moderate inhibition of ectoapyrase activity by application of low concentrations of chemical apyrase inhibitors would cause naturally occurring levels of eATP to increase resulting in stomatal opening. We found that, similar to treatment with 15 μM ATPγS, treatment of leaves with two different apyrase inhibitors at a concentration of 1.5 µg/mL also induces stomatal opening (Fig. 1A).We Keywords: extracellular ATP, apyrase, stomata, abscisic acid, ArabidopsisAbbreviations: ABA, abscisic acid; eATP, extracellular ATP; ROS, reactive oxygen species; NO, nitric oxide; RBOHD/F, NADHP oxidase homolog sub-units D/F; DTT, dithiothreitol; DPI, diphenyleneiodonium; GCA2, growth controlled by ABA 2; ABI2, Abscisic Acid-Insensitive2; PA, phosphatidic acid; gpa, α-subunit of the Arabidopsis heterotrimeric G Protein in Arabidopsis leaves there is a bi-phasic dose-response to applied nucleotides; i.e., lower concentrations induce stomatal opening, while higher concentrations induce closure. two mammalian purinoceptor antagonists, PPaDS and rB2, block both nucleotide-induced stomatal opening and closing. these antagonists also partially block aBa-induced stomatal closure and light-induced stomatal opening. there are two closely related Arabidopsis apyrases, ataPY1 and ataPY2, which are both expressed in guard cells. here we report that low levels of apyrase chemical inhibitors can induce stomatal opening in the dark, while apyrase enzyme blocks aBa-induced stomatal closure. We also demonstrate that high concentrations of atP induce stomatal closure in the light. application of atPγS and chemical apyrase inhibitors at concentrations that have no effect on stomatal closure can lower the threshold for aBa-induced closure. the closure induced by atPγS was not observed in gpa1-3 loss-of-function mutants. these results further confirm the role of extracellular atP in regulating stomatal apertures.

Extracellular ATP mediates H 2 S-regulated stomatal movements and guard cell K + current in a H 2 O 2 -dependent manner in Arabidopsis

“…The eATP regulates stomatal movement, and the regulation is a biphasic dose-response process, namely eATP promotes stomatal opening when the concentration of eATP below 30 lmol/L; on the contrary, it causes stomatal closure when its concentration is higher than 150 lmol/L [22]. This hypothesis was further confirmed by the experiments of Clark et al [23]; they found that low levels of apyrase chemical inhibitors can induce stomatal opening in the dark, while apyrase enzyme blocks ABA-induced stomatal closure. At present, the relationship between H 2 S and eATP is unknown, and it is also unclear how eATP originates in guard cells.…”

The Biochemical Properties of the Arabidopsis Ecto-Nucleoside Triphosphate Diphosphohydrolase AtAPY1 Contradict a Direct Role in Purinergic Signaling

“…), belonging to the structural class of sulfonamides. Although these inhibitors turned out not to be completely apyrase-specific because they inhibited alkaline phosphatases as well [ 61 ], they have become useful tools in deciphering the physiological role of extracellular E-NTPDases in plants [ 23 , 24 , 29 , 62 – 64 ].…”

Extracellular ATP elicits DORN1-mediated RBOHD phosphorylation to regulate stomatal aperture

“…The addition of ATP was previously shown to trigger ROS production 9 , 19 . Independent reports also indicated that ATP addition could induce closure of stomata 6 , 59 . However, in neither case was a mechanism provided for these phenomena.…”