Identification of a Plant Nitric Oxide Synthase Gene Involved in Hormonal Signaling

Nitric oxide (NO) functions as a signal and a cytotoxic agent in many physiological and immunological processes in animals (1–3) and is synthesized by nitric oxide synthase (NOS) (2, 4–6). There are three known isoforms of NOS [neuronal (nNOS), inducible (iNOS), and endothelial (eNOS)], and each contains a heme-oxygenase and a flavin reductase domain. Related NOS genes have been described in many eukaryotic species from vertebrates to fungi (6, 7). Bacteria also encode NOS proteins that are smaller, containing only the oxygenase domain, which is similar in sequence and biochemical activity to the mammalian NOS oxygenase (8–10).

In plants, NO serves as a signal in hormonal and defense responses (3, 7, 11–14). The source of NO synthesis in plants has been the subject of much debate and includes reduction of nitrite by nitrate reductase (NR) and the oxidation of arginine to citrulline by NOS (3, 7, 13, 15). NOS has been implicated in plants by the detection of NOS activity that can be inhibited by mammalian NOS inhibitors; cross-reactivity of plant proteins to mammalian antibody to NOS has also been detected (3, 13–15). However, a recent proteomic analysis showed that such cross-reacting proteins were unrelated to NOS (16). No gene or protein with sequence similarity to known mammalian-type NOS has been found in plants (15, 16). These findings suggest that plants have a different NOS enzyme. This has been confirmed by the recent discovery of a plant NOS gene that is induced by viral infection and encodes a variant of the P protein of glycine decarboxylase (GDC) (17).

An Arabidopsis gene, which we name AtNOS1 (18), encodes a protein (fig. S1) with sequence similarity to a protein that has been implicated in NO synthesis in the snail Helix pomatia (19). A homozygous mutant line with a DNA insertion in the first exon of this gene was isolated (fig. S2). Leaf extracts were assayed for NOS activity (formation of [³H]-citrulline from L-[³H]-arginine). NOS activity in the mutant was 25% that of wild type (Fig. 1A). To verify that the wild-type activity was due to a NOS-like enzyme, inhibition by the NOS inhibitor N^G-nitro-L-Arg-methyl ester (L-NAME) was demonstrated (Fig. 1A).

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Endogenous NO production was examined in roots by comparing wild-type and mutant seedlings loaded with the permeable NO-sensitive dye fluorophore 4,5-diaminofluorescein diacetate (DAF-2 DA). NO production, which is strongest at the root tip of young seedlings, was greatly diminished in the mutant (Fig. 1, C and D). When roots were treated with 50 μM abscisic acid (ABA), NO levels increased in the wild-type plants but were still much lower in the mutant (Fig. 1, E and F). ABA-induced NO production was inhibited by L-NAME (Fig. 1, G and H) but did not coincide with a significant increase in AtNOS1 mRNA (Fig. 1B). These results demonstrate that AtNOS1 is involved in NO synthesis in Arabidopsis.

To determine whether AtNOS1 encodes an enzyme that has NOS activity, the AtNOS1 protein was expressed in bacteria as a fusion protein with glutathione-S-transferase (GST-AtNOS1), then purified and assayed. An AtNOS1 cDNA clone was isolated and found to encode a protein of 561 amino acid residues (fig. S1). Extracts from bacteria expressing the fusion protein showed enhanced levels of NOS activity (Fig. 2A). The protein, purified 80-fold by glutathione-affinity chromatography, showed NOS activity dependent on nicotinamide adenine dinucleotide phosphate, calmodulin, and Ca²⁺ and inhibited by L-NAME (Fig. 2B), properties similar to mammalian eNOS and nNOS. The activity was not stimulated by tetrahydrobiopterin (BH₄), flavin adenine dinucleotide (FAD), riboflavin 5′-phosphate (FMN) (Fig. 2B), or heme (Fig. 2D), which are all cofactors of mammalian NOS. AtNOS1 could use the mammalian NOS intermediate N-hydroxyarginine (NOHA) as a substrate (Fig. 2D). Kinetic analysis using the Greiss reagent to measure NO production revealed a K_m of 12.5 μM for arginine and a maximum velocity (V_max) of 5.0 nmole min^–1 mg^–1 (Fig. 2C). These experiments show that AtNOS1 encodes a plant NOS with several distinct properties.

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The phenotype of Atnos1 mutant plants was examined to determine the role of AtNOS1 in plant growth. During the early stages of development, the first true leaves of the Atnos1 mutants failed to fully green (Fig. 3, A and B). More mature mutants showed reduced shoot (Fig. 3D), root (Fig. 3E), and inflorescence (Fig. 3F) growth, and fertility was low. We verified that the shoot phenotypes cosegregate with the Atnos1 mutation by backcrossing the mutant to wild-type plants; all 21 F2 mutant plants examined were homozygous for the DNA insertion as determined by polymerase chain reaction (PCR)–based genotyping (20).

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Genetic complementation showed that poor leaf greening and shoot growth could be reversed by expressing the AtNOS1 cDNA with a viral promoter (the CaMV 35S promoter) in Atnos1 mutants (Fig. 3, C and D). Western blots, with polyclonal antisera against the 19 N-terminal amino acids of AtNOS1, confirmed the restoration of AtNOS1 expression in rescued lines (Fig. 3G). NOS activity in leaf extracts was also restored to levels higher than those found in wild-type extracts (Fig. 1A). When Atnos1 mutant plants were treated with 100 μM sodium nitroprusside (SNP, an NO donor), greening, growth, and fertility were restored (Fig. 3, H to J) (20), indicating that these phenotypes were due to a deficiency in NO production caused by the Atnos1 mutation. Control treatments with sodium ferrocyanide had no effect (Fig. 3K).

The phytohormone ABA enhances NO synthesis in and induces closure of stomates (21–23). Both NR and NOS have been implicated in these processes (15, 21, 23). To address the role of AtNOS1 in stomates, NO production and stomatal closure in epidermal peels from wild-type and Atnos1 plants were compared. ABA-induced NO production (measured as increased fluorescence of the NO-sensitive dye DAF-2 DA) was severely inhibited in Atnos1 guard cells compared with wild-type and rescued (expressing AtNOS1) mutant lines (Fig. 4, A and B). L-NAME at 200 μM inhibited ABA-induced NO production (Fig. 4, A and B). In stomatal closure experiments, ABA elicited little response from Atnos1 stomates (12% closure at 50 μM ABA) compared with wild-type and rescued mutant stomates (58% and 41% closure for wild-type and rescued mutants, respectively) (Fig. 4C). ABA also failed to inhibit light-induced stomatal opening in Atnos1 mutants under conditions in which wild-type and rescued Atnos1 stomates were strongly inhibited by ABA (Fig. 4D). These results show that AtNOS1 is required for ABA-induced NO generation and stomatal closure and further indicate that AtNOS1-dependent NO generation occurs downstream of ABA in this signaling pathway.

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In plants, the source(s) of NO production and their importance have been the subject of much debate. The identification of plant NOS genes demonstrates that NOS enzymes exist in plants. There are several striking similarities between plant and mammalian NOS enzymes. The plant iNOS protein (17) has a high V_max for NO production and is inducible like mammalian iNOS. AtNOS1 resembles nNOS and eNOS in that it is constitutively expressed, is stimulated by Ca²⁺ and CaM, and has a lower output of NO. This resemblance has limits, however, as the plant iNOS is regulated by added Ca²⁺ (17), unlike the mammalian iNOS, and mammalian eNOS and nNOS genes are inducible by certain stimuli (24); AtNOS1's response has only been examined for ABA. Most important, the sequences of the plant NOS proteins are not similar to those of their mammalian counterparts. iNOS resembles a component of GDC, and AtNOS1 is most similar to a group of hypothetical bacterial proteins that have putative GTP-binding or GTPase domains, as noted for the H. pomatia protein (19). Further investigation into the structure and function of the plant NOS proteins is needed to clarify the mechanisms of their action. The mutation in AtNOS1, however, demonstrates that this gene plays a vital role in plant growth, fertility, stomatal movements, and hormone signaling.