Evidence supporting the existence of l ‐arginine‐dependent nitric oxide synthase activity in plantsписьмо
Аннотация: The important physiological significance of the gaseous free radical nitric oxide (NO) in living beings is acknowledged worldwide. However, understanding the biological implications of NO has not been an easy task. In 1960, Fewson & Nicholas published a brief report indicating that microorganisms and higher plants could use NO as an intermediate in nitrogen metabolism. Nineteen years later, Keppler (1979) reported that plants can generate this molecule. However, it was not until 1987, with the identification of NO as the endothelium-derived relaxing factor (EDRF) (Palmer et al., 1987), when the fundamentals of a new research area were established, thus generating a boost to investigations on NO in biology. At present, research on NO is still increasing, with c. 96 000 papers found in the PubMed database. Nitric oxide can be produced in plants by nonenzymatic and enzymatic systems (del Río et al., 2004). There are several enzymatic systems that have been shown to produce NO, mainly including nitrate reductase (Rockel et al., 2002) and nitrite reductase (Stöhr et al., 2001) as a side-reaction, a mitochondrial-dependent nitrite-reducing activity (Modolo et al., 2005; Planchet et al., 2005) and l-arginine-dependent nitric oxide synthase (NOS) activity (Barroso et al., 1999; Corpas et al., 2004; del Río et al., 2004). In animal systems, after the identification of NO as a molecule of physiological importance, intensive research was carried out in multiple laboratories to identify the endogenous source of NO. As a result, a new enzyme with different molecular forms, designated as NOS, which generated NO and citrulline from the amino acid l-arginine, was discovered (Moncada et al., 1989). By comparison, research on NO in plants has advanced more slowly, although at present it is widely admitted that this radical is endogenously generated in plant cells. Perhaps the main reason for this sluggishness has been the failure to carry out the molecular characterization of the enzymatic NO source(s), which is still an important challenge for plant biologists (Zemojtel et al., 2006; Moreau et al., 2008). Therefore, in plant cells there is a pressing need to know which enzymatic system is responsible for l-arginine-dependent NO generation and its intracellular localization. The response to these questions would allow investigations, using appropriate mutants, to be carried out regarding the modulation and control of NO biosynthesis in plants under normal physiological conditions and under different biotic and abiotic stress situations. The enzymatic activity of mammalian NOS is a remarkably complex reaction. This homodimeric enzyme contains a heme group as part of its catalytic site and requires several cofactors, including NADPH, FAD, FMN, calcium, calmodulin and tetrahydrobiopterin (BH4), to generate NO and citrulline from l-arginine in the presence of oxygen (Alderton et al., 2001). In this reaction, a small electron transport chain is involved where a total of five electrons are transferred in the direction NADPH→ FAD→FMN, with the heme iron being the final acceptor. In three different plant species, the biochemical characterization of the cofactor requirements of NOS activity has shown that this activity requires the same cofactors as the mammalian NOS isoforms (Table 1). Examples of the characterization of NOS activity from plant origin using the ozone chemiluminescence assay are also shown in Fig. 1(a). It is interesting to point out that mammalian NOS requires BH4, which seems to promote and/or stabilize the active dimeric form of the mammalian NOS isoforms (Alderton et al., 2001). However, in higher plants, the presence of BH4 is not clear, and its function could perhaps be carried out by tetrahydrofolate (FH4) whose biosynthesis and distribution is well known in higher plants (Sahr et al., 2005). However, further research is necessary to clarify this point. Biochemical characterization of l-arginine-dependent nitric oxide synthase (NOS) activity (a) and confocal laser-scanning microscopy (CLSM) detection and visualization of endogenous nitric oxide (NO) in plant leaves (b–d). (a) Biochemical characterization of l-arginine-dependent NOS activity in olive leaves and peroxisomes isolated from pea leaves. Reaction mixtures containing plant samples were incubated in the absence and presence of l-arginine (1 mm or 0.1 mm), NADPH (1 mm), EGTA (0.5 mm), cofactors (10 µm FAD, 10 µm FMN and 10 µm BH4), 1 mm aminoguanidine (AG, an animal NOS activity inhibitor), antibody against animal iNOS (Anti-iNOS), carboxymethoxylamine (CM) and aminoacetonitrile (AAN), two inhibitors of the P protein of the glycine decarboxylase, or azide (an inhibitor of nitrate reductase). Other reaction mixtures were pre-incubated at 95°C for 10 min. The NOS activity was quantified from the NO produced, which was determined by the ozone chemiluminescence method. l-Arg, l-arginine. (Left panel from Valderrama et al., 2007; right panel from Corpas et al., 2004.). (b) Cross-section of pea leaf incubated with DAF-FM DA (4-aminomethyl-2′,7′-difluorofluorescein diacetate) (10 µm) used as a fluorescent probe to detect NO. (c) Cross-section of pea leaf pre-incubated for 30 min with 200 µm PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl) (a NO scavenger) and then incubated with DAF-FM DA. (d) Cross-section of pea leaf pre-incubated 60 min with 5 mm NG-nitro-l-arginine methyl ester (l-NAME) (a NOS activity inhibitor) and then incubated with DAF-FM DA. Each picture was prepared from at least 20 cross-sections analyzed by CLSM. Strong and bright green fluorescence corresponds to NO. The red–orange colour corresponds to the chlorophyll autofluorescence. E1, adaxial epidermis; E2, abaxial epidermis; Pm, palisade mesophyll; Sm, spongy mesophyll; V, main vein. Bar, 100 µm (from Corpas et al., 2008b). A summary of the l-arginine-dependent NOS activity detected in tissues of different plant species using complementary techniques that have been previously validated and employed in animal systems is shown in Table 2. These techniques include the arginine-citrulline assay, ozone chemiluminescence, spin trapping electron paramagnetic resonance (EPR), and the commercial NOS assay kit. Each of these methods has advantages and disadvantages that are mainly related to the specificity, sensitivity and/or complexity of the different techniques (for a review see Yao et al., 2004). However, spin trapping EPR and ozone chemiluminescence methods provide reliable measurements of NO in biological samples in the range of nanomolar and picomolar concentrations (Archer, 1993) and these methods have also been used in plant samples (Corpas et al., 2004, 2008a; Jasid et al., 2006). However, although the arginine-citrulline assay has been widely used, its validity has recently been questioned in Arabidopsis extracts as a result of the interference of an enzyme of the urea cycle, which, using l-arginine as a substrate, produces a product other than citrulline (Tischner et al., 2007). In any case, from the data presented in Table 2 it can be concluded that l-arginine-dependent NOS activity is present bona fide in plant cells and is localized in at least two subcellular compartments, including peroxisomes (Barroso et al., 1999; Corpas et al., 2004) and chloroplasts (Jasid et al., 2006). The presence of NOS activity in plant peroxisomes was extended, years later, to animal peroxisomes (Stolz et al., 2002; Loughran et al., 2005). There is also EPR evidence of the generation of NO in peroxisomes (Corpas et al., 2004) and chloroplasts (Jasid et al., 2006) isolated from pea and soybean leaves, respectively. Moreover, it has been suggested that peroxisomes could be the source of NO implicated in growth regulation and re-orientation of pollen tubes in Lilium longiflorum (Prado et al., 2004). More recently, genetic approaches in Arabidopsis plants on the protein now designated as nitric oxide-associated protein 1 (NOA1), using loss-of-function mutants, have suggested that chloroplasts of this plant species could also be involved in NO generation (Gas et al., 2009). However, it is interesting to note that in plants, in contrast to the situation in mammalian tissues (Tatoyan & Giulivi, 1998), the occurrence of l-arginine-dependent NOS activity has not been detected in mitochondria (Barroso et al., 1999). In confocal laser-scanning microscopy experiments using the fluorescent probe DAF-FM DA (4-aminomethyl-2′,7′-difluorofluorescein diacetate), the occurrence of NO in mitochondria from Arabidopsis leaves was detected (Guo & Crawford, 2005). However, the source of this NO does not seem to be a NOS activity because the previous candidates for plant NOS enzymes have been shown to either not exist (iNOS) or to be a GTPase (AtNOS1) without l-arginine-dependent NOS activity (Guo et al., 2003; Klessig et al., 2004; Guo & Crawford, 2005; Zemojtel et al., 2006; Crawford et al., 2006; Moreau et al., 2008). The NO detected in mitochondria from Arabidopsis leaves could be produced by reduction of in the mitochondrial electron transport chain, as previously reported by Modolo et al. (2005). In plant tissues, a significant number of reports using physiological and/or pharmacological approaches with inhibitors analogous to l-arginine, such as NG-nitro-l-arginine methyl ester (l-NAME), NG-nitro-l-arginine (l-NNA) or l-NG-monomethyl-arginine monoacetate (l-NMMA), have shown a decrease in NO production, thus corroborating the involvement of an l-arginine-dependent NOS activity in the generation of NO (Fig. 1b–d). In addition, these reductions in NO generation affected some physiological processes in the plants investigated. Some examples of this are discussed. (1) In Vicia faba, pretreatment of the whole plant with l-NAME substantially suppressed ultraviolet B (UVB)-induced and hydrogen peroxide (H2O2)-induced stomatal closure and NO generation, indicating that guard cells possess a NOS-like enzyme that generates NO in response to UVB radiation (Neill et al., 2002, 2008a; He et al., 2005). (2) During the Arabidopsis hypersensitive response, NO synthesis was blocked in the presence of l-NNA (Delledonne et al., 1998) and l-NMMA (Zhang et al., 2003). Additionally, when Arabidopsis thaliana cells interacted with lipopolysaccharide (LPS; components of gram-negative bacteria that stimulate the inducible NOS in animal cells), a rapid burst of NO was provoked. This NO production was reduced dramatically by l-NNA; however, it was not affected by sodium azide, a potent inhibitor of nitrate reductase (NR), indicating that NR is not involved in NO synthesis in this situation (Zeidler et al., 2004). Furthermore, LPS activation of NO generation was also blocked by l-NAME, a calmodulin (CaM) antagonist, and by a Ca2+ chelator, indicating the involvement of NOS activity, which is dependent on calcium and CaM (Ali et al., 2007; Ma et al., 2008). (3) In Arabidopsis roots, ABA-induced NO production is inhibited by l-NAME (Guo et al., 2003). (4) In pea plants the l-arginine-dependent NOS activity of isolated leaf peroxisomes is strongly down-regulated by senescence (Corpas et al., 2004). (5) In Hibiscus moscheutos, root elongation is markedly reduced by treatment with l-NNA (Tian et al., 2007). (6) In Taxus cuspidata cells, NO production is substantially reduced after incubation with l-NNA (Xiao et al., 2009). (7) In Salvia miltiorrhiza hairy roots, ATP-induced NO production is significantly suppressed by l-NAME (Wu & Wu, 2008). (8) Finally, in maize leaves, l-NAME partially blocked UV-B-induced NO accumulation (Tossi et al., 2009). Besides, the screening of Arabidopsis using several strategies has also provided genetic evidence for the presence of an l-arginine-dependent NOS activity in plants. For instance, the discovery of several mutants with an imbalance in the l-arginine content, which affects the generation of NO, can be considered as indirect evidence for the presence of NOS activity. The mutant designated as nox1 was characterized by an increased production of NO and correlated well with the accumulation of l-arginine (He et al., 2004). More recently, Arabidopsis mutants knockout for the enzyme arginase (which decomposes l-arginine to urea and ornithine) brought about an accumulation of l-arginine in seedlings, which was accompanied by an increase in the production of NO and in the formation of lateral and adventitious roots (Flores et al., 2008). A gene or a protein with homology to mammalian NOS enzymes has not been found in Arabidopsis (The Arabidopsis genome initiative, 2000). The different molecular approaches developed so far to clone a plant NOS, based on the sequence of animal NOS, have always given negative results and this has been a source of considerable frustration in researchers (Zemojtel et al., 2006; Neill et al., 2008b; Wilson et al., 2008; Gas et al., 2009). However, the set of data available from at least 11 different plant species are strong enough to support the existence of NOS activity in plants. Considering all the results available, it is clear that the plant NOS is not a canonical animal NOS enzyme and that perhaps, in plant cells, NOS activity is carried out by several proteins that could function together to generate NO from l-arginine, but, strikingly, using the same substrate and cofactors as the animal NOS. Nevertheless, the possibility of the presence of an enzyme that generates NO from l-arginine as a residual by-product cannot be rejected. Therefore, using all the biochemical, genetic, physiological and pharmacological information obtained, considerable efforts need to be made to elucidate the identity of the plant l-arginine-dependent NOS activity. The purification of l-arginine-dependent NOS activity from isolated peroxisomes of pea leaves is underway in our laboratory. After a first purification step using anion-exchange Fast Protein Liquid Chromatography, a single peak of l-arginine-dependent NOS activity was obtained, although the protein yields were extremely low. This could indicate that either plant NOS is a single protein or that the components of the possible enzymatic complex have a similar charge. The availability of more information on the cell localization of plant NOS will also contribute to our understanding of the cross-talk between cell organelles and its involvement in signalling processes. The identification and molecular characterization of the enzyme responsible for this activity in plants will be a great challenge for the forthcoming years. Thus, the search of the Holy Grail, as the identification of the plant NOS was previously described by Wendehenne et al. (2003), still continues. We thank Dr Manuel Rodríguez-Concepción (Centre for Research on Agricultural Genomics, Barcelona, Spain) for his comments. This work was supported by grants from the Ministry of Education and Science (BIO2006-14949-C02-01 and BIO2006-14949-C02-02) and Junta de Andalucía (project P06-CVI-1820), Spain.
Год издания: 2009
Издательство: Wiley
Источник: New Phytologist
Ключевые слова: Plant Stress Responses and Tolerance, Photosynthetic Processes and Mechanisms, Nitric Oxide and Endothelin Effects
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Том: 184
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Страницы: 9–14