Lorenzo Lamattina

Nitric oxide stimulates seed germination and de-etiolation, and inhibits hypocotyl elongation, three light-inducible responses in plants.

Abstract. Seed germination, greening of etiolated plants and inhibition of hypocotyl elongation are stimulated by light, which is sensed by various types of photoreceptor. Nitric oxide (NO) has proven to be a bioactive molecule, especially in mammalian cells and, most recently, in plants. Like some phytochrome-dependent processes, many NO-mediated ones are accomplished through increases in cGMP levels. Given these similarities, we proposed that NO could take part in light-mediated events in plants. Here we show that NO promotes seed germination and de-etiolation, and inhibits hypocotyl and internode elongation, processes mediated by light. Two NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine induced germination of lettuce (Lactuca sativa L. cv. Grand Rapids) seeds in conditions in which this process is dependent on light (e.g. 26 °C). This was a dose-dependent response and was arrested by addition of an NO scavenger, carboxy-PTIO. In addition, nitrite and nitrate, two NO-decomposition products were ineffective in stimulating germination. Wheat seedlings sprayed with SNP and grown in darkness contained 30–40% more chlorophyll than control seedlings. Nitric-oxide-mediated partial greening was increased by light pulses, wounding and biotic stress. Arabidopsis thaliana (L.) Heynh. (ecotype Columbia) and lettuce seedlings grown in the dark had 20%-shorter hypocotyls in NO treatments than in control ones. On the other hand, internode lengths of potato plants growing under low light intensity and sprayed with 100 μM SNP were also 20% shorter than control ones. These results implicate NO as a stimulator molecule in plant photomorphogenesis, either dependent on or independent of plant photoreceptors.

Nitric Oxide Is Required for Root Organogenesis

In this report, we demonstrate that nitric oxide (NO) mediates the auxin response leading the adventitious root formation. A transient increase in NO concentration was shown to be required and to be part of the molecular events involved in adventitious root development induced by indole acetic acid

Nitric oxide regulates K+ and Cl- channels in guard cells through a subset of abscisic acid-evoked signaling pathways

Abscisic acid (ABA) triggers a complex sequence of signaling events that lead to concerted modulation of ion channels at the plasma membrane of guard cells and solute efflux to drive stomatal closure in plant leaves. Recent work has indicated that nitric oxide (NO) and its synthesis are a prerequisite for ABA signal transduction in Arabidopsis and Vicia guard cells. Its mechanism(s) of action is not well defined in guard cells and, generally, in higher plants. Here we show directly that NO selectively regulates Ca2+-sensitive ion channels of Vicia guard cells by promoting Ca2+ release from intracellular stores to raise cytosolic-free [Ca2+]. NO-sensitive Ca2+ release was blocked by antagonists of guanylate cyclase and cyclic ADP ribose-dependent endomembrane Ca2+ channels, implying an action mediated via a cGMP-dependent cascade. NO did not recapitulate ABA-evoked control of plasma membrane Ca2+ channels and Ca2+-insensitive K+ channels, and NO scavengers failed to block the activation of these K+ channels evoked by ABA. These results place NO action firmly within one branch of the Ca2+-signaling pathways engaged by ABA and define the boundaries of parallel signaling events in the control of guard cell movements.

Nitric Oxide Acts as an Antioxidant and Delays Programmed Cell Death in Barley Aleurone Layers

Nitric oxide (NO) is a freely diffusible, gaseous free radical and an important signaling molecule in animals. In plants, NO influences aspects of growth and development, and can affect plant responses to stress. In some cases, the effects of NO are the result of its interaction with reactive oxygen species (ROS). These interactions can be cytotoxic or protective. Because gibberellin (GA)-induced programmed cell death (PCD) in barley (Hordeum vulgare cv Himalaya) aleurone layers is mediated by ROS, we examined the effects of NO donors on PCD and ROS-metabolizing enzymes in this system. NO donors delay PCD in layers treated with GA, but do not inhibit metabolism in general, or the GA-induced synthesis and secretion of α-amylase. α-Amylase secretion is stimulated slightly by NO donors. The effects of NO donors are specific for NO, because they can be blocked completely by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The antioxidant butylated hydroxy toluene also slowed PCD, and these data support our hypothesis that NO is a protective antioxidant in aleurone cells. The amounts of CAT and SOD, two enzymes that metabolize ROS, are greatly reduced in aleurone layers treated with GA. Treatment with GA in the presence of NO donors delays the loss of CAT and SOD. We speculate that NO may be an endogenous modulator of PCD in barley aleurone cells.

Nitric Oxide and Cyclic GMP Are Messengers in the Indole Acetic Acid-Induced Adventitious Rooting Process

This report describes part of the signaling pathway and some of the molecules involved in the auxin-induced adventitious root formation in cucumber (Cucumis sativus). Previous results showed that nitric oxide (NO) mediates the auxin response during adventitious root formation (Pagnussat et al., 2002). To determine the order of action of indole acetic acid (IAA) and NO within the signal transduction pathway and to elucidate the target molecules that are downstream of NO action, cucumber hypocotyl cuttings were submitted to a pretreatment leading to endogenous auxin depletion. The auxin depletion treatment provoked a 3-fold reduction of the root number in comparison to the nondepleted explants. The NO-donor sodium nitroprusside was able to promote adventitious rooting in auxin-depleted explants, whereas the specific NO scavenger cPTIO prevented the effect of sodium nitroprusside. The endogenous NO level was monitored in both control and auxin-depleted explants using a NO-specific fluorescent probe. The NO level was 3.5-fold higher in control (nondepleted) explants than in auxin-depleted ones. The exogenous application of IAA restored the NO concentration to the level found in nondepleted explants. Because NO activates the enzyme guanylate cyclase (GC), we analyzed the involvement of the messenger cGMP in the adventitious root development mediated by IAA and NO. The GC inhibitor LY83583 reduced root development induced by IAA and NO, whereas the cell-permeable cGMP derivative 8-Br-cGMP reversed this effect. The endogenous level of cGMP is regulated by both the synthesis via GC and its degradation by the phosphodiesterase activity. When assayed, the phosphodiesterase inhibitor sildenafil citrate was able to induce adventitious rooting in both nondepleted and auxin-depleted explants. Results indicate that NO operates downstream of IAA promoting adventitious root development through the GC-catalyzed synthesis of cGMP.

Nitric Oxide and Abscisic Acid Cross Talk in Guard Cells

Despite recent efforts to elucidate the regulation of stomatal movement, many components within the branched pathways of guard cell abscisic acid (ABA) signaling remain to be identified. Here, we show results supporting the involvement of nitric oxide (NO) as a new component of this signaling

Nitric oxide counteracts cytotoxic processes mediated by reactive oxygen species in plant tissues

Abstract. Many environmental conditions subject plants to oxidative stress, in which reactive oxygen species (ROS) are overproduced. These ROS act as transduction signals in plant defense responses, but also cause effects that result in cellular damage. Since nitric oxide (NO) is a bioactive molecule able to scavenge ROS, we analyzed its effect on some cytotoxic processes produced by ROS in potato (Solanum tuberosum L. cv. Pampeana) leaves. Two NO donors: (i) sodium nitroprusside and (ii) a mixed solution of ascorbic acid and NaNO2, were able to prevent chlorophyll loss mediated by the methyl viologen herbicide diquat (a ROS generator), with effective concentrations falling between 10 and 100 μM of the donors. This protection was mimicked by thiourea and penicillamine, two antioxidant compounds. Residual products from NO generation and decomposition failed to prevent chlorophyll decline. A specific NO scavenger, the potassium salt of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), arrested NO-mediated chlorophyll protection. In addition, some events mediated by ROS during infection of potato leaves with Phytophthora infestans (race 1, 4, 7, 8, 10, 11, mating type A2) were also examined. In this sense, NO proved to markedly decrease ion leakage and the number of lesions, indicative of cell death, produced upon infection in potato leaves. The NO-mediated decrease in ion leakage was also inhibited by carboxy-PTIO. Fragmentation of DNA diminished when P. infestans-infected potato leaves were treated with 100 μM SNP. These results suggest that, acting as an antioxidant, NO can strongly counteract many ROS-mediated cytotoxic processes in plants. Moreover, the evidence of NO functionality in the plant kingdom is strengthened by this work.

Nitric Oxide Mediates the Indole Acetic Acid Induction Activation of a Mitogen-Activated Protein Kinase Cascade Involved in Adventitious Root Development

Recently, it was demonstrated that nitric oxide (NO) and cGMP are involved in the auxin response during the adventitious rooting process in cucumber (Cucumis sativus; Pagnussat et al., 2002, 2003). However, not much is known about the complex molecular network operating during the cell proliferation and morphogenesis triggered by auxins and NO in that process. Anatomical studies showed that formation of adventitious root primordia was clearly detected in indole acetic acid (IAA)- and NO-treated cucumber explants, while neither cell proliferation nor differentiation into root primordia could be observed in control explants 3 d after primary root was removed. In order to go further with signal transduction mechanisms that operate during IAA- and NO-induced adventitious root formation, experiments were designed to test the involvement of a mitogen-activated protein kinase (MAPK) cascade in that process. Cucumber explants were treated with the NO-donor sodium nitroprusside (SNP) or with SNP plus the specific NO-scavenger cPTIO. Protein extracts from those explants were assayed for protein kinase (PK) activity by using myelin basic protein (MBP) as substrate in both in vitro and in-gel assays. The activation of a PK of approximately 48 kD could be detected 1 d after NO treatment with a maximal activation after 3 d of treatment. In control explants, a PK activity was detected only after 4 d of treatment. The MBP-kinase activity was also detected in extracts from IAA-treated explants, while no signal was observed in IAA + cPTIO treatments. The PK activity could be inhibited by the cell-permeable MAPK kinase inhibitor PD098059, suggesting that the NO-dependent MBP-kinase activity is a MAPK. Furthermore, when PD098059 was administered to explants treated with SNP or IAA, it produced a delay in root emergence and a dose-dependent reduction in root number. Altogether, our results suggest that a MAPK signaling cascade is activated during the adventitious rooting process induced by IAA in a NO-mediated but cGMP-independent pathway. The activation of MAPKs is discussed in relation to the cell responses modulating mitotic process.

Nitric Oxide Improves Internal Iron Availability in Plants

Iron deficiency impairs chlorophyll biosynthesis and chloroplast development. In leaves, most of the iron must cross several biological membranes to reach the chloroplast. The components involved in the complex internal iron transport are largely unknown. Nitric oxide (NO), a bioactive free radical, can react with transition metals to form metal-nitrosyl complexes. Sodium nitroprusside, an NO donor, completely prevented leaf interveinal chlorosis in maize (Zea mays) plants growing with an iron concentration as low as 10 μmFe-EDTA in the nutrient solution.S-Nitroso-N-acetylpenicillamine, another NO donor, as well as gaseous NO supply in a translucent chamber were also able to revert the iron deficiency symptoms. A specific NO scavenger, 2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, blocked the effect of the NO donors. The effect of NO treatment on the photosynthetic apparatus of iron-deficient plants was also studied. Electron micrographs of mesophyll cells from iron-deficient maize plants revealed plastids with few photosynthetic lamellae and rudimentary grana. In contrast, in NO-treated maize plants, mesophyll chloroplast appeared completely developed. NO treatment did not increase iron content in plant organs, when expressed in a fresh matter basis, suggesting that root iron uptake was not enhanced. NO scavengers 2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and methylene blue promoted interveinal chlorosis in iron-replete maize plants (growing in 250 μm Fe-EDTA). Even though results support a role for endogenous NO in iron nutrition, experiments did not establish an essential role. NO was also able to revert the chlorotic phenotype of the iron-inefficient maize mutants yellow stripe1 and yellow stripe3, both impaired in the iron uptake mechanisms. All together, these results support a biological action of NO on the availability and/or delivery of metabolically active iron within the plant.

Nitric oxide function in plant biology: a redox cue in deconvolution

Nitric oxide (NO), a gaseous, redox-active small molecule, is gradually becoming established as a central regulator of growth, development, immunity and environmental interactions in plants. A major route for the transfer of NO bioactivity is S-nitrosylation, the covalent attachment of an NO moiety to a protein cysteine thiol to form an S-nitrosothiol (SNO). This chemical transformation is rapidly emerging as a prototypic, redox-based post-translational modification integral to the life of plants. Here we review the myriad roles of NO and SNOs in plant biology and, where known, the molecular mechanisms underpining their activity.