A new role for an old enzyme: Nitrate reductase-mediated nitric oxide generation is required for abscisic acid-induced stomatal closure in <i>Arabidopsis</i> <i>thaliana</i>

TLDR

Abscisic acid triggers stomatal closure through complex signaling cascades, with nitric oxide identified as a key mediator, yet the cellular source of NO in guard cells remains unknown. This study investigates whether nitrate reductase (NR) is the enzyme responsible for NO synthesis in guard cells during ABA‑induced stomatal closure. Using pharmacological inhibition, physiological assays, and genetic mutants (wild‑type, NR double mutant nia1, nia2, and ABA‑insensitive abi1‑1/abi2‑1), the authors demonstrate that NR activity generates NO in response to ABA and nitrite and that ABI1/ABI2 phosphatases act downstream of NO. The data show that NR‑mediated NO production is essential for ABA‑induced stomatal closure, as NR mutants fail to produce NO and close stomata, while ABI1/ABI2 mutants place NO downstream in the signaling cascade.

Abstract

The plant hormone abscisic acid (ABA), synthesized in response to water-deficit stress, induces stomatal closure via activation of complex signaling cascades. Recent work has established that nitric oxide (NO) is a key signaling molecule mediating ABA-induced stomatal closure. However, the biosynthetic origin of NO in guard cells has not yet been resolved. Here, we provide pharmacological, physiological, and genetic evidence that NO synthesis in Arabidopsis guard cells is mediated by the enzyme nitrate reductase (NR). Guard cells of wild-type Arabidopsis generate NO in response to treatment with ABA and nitrite, a substrate for NR. Moreover, NR-mediated NO synthesis is required for ABA-induced stomatal closure. However, in the NR double mutant, nia1, nia2 that has diminished NR activity, guard cells do not synthesize NO nor do the stomata close in response to ABA or nitrite, although stomatal opening is still inhibited by ABA. Furthermore, by using the ABA-insensitive (ABI) abi1-1 and abi2-1 mutants, we show that the ABI1 and ABI2 protein phosphatases are downstream of NO in the ABA signal-transduction cascade. These data demonstrate a previously uncharacterized signaling role for NR, that of mediating ABA-induced NO synthesis in Arabidopsis guard cells.