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Keywords:

  • Nicotiana tabacum;
  • Nicotiana plumbaginifolia;
  • abiotic stress;
  • nitrogen radicals;
  • reactive oxygen species

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

The function of nitric oxide (NO), a gaseous free radical emitted by many plants, is incompletely understood. In the present study the hypothesis that NO generation, like that of the reactive oxygen species, occurs as a general response to different environmental cues was tested. Leaf peels and mesophyll cell suspensions of Nicotiana tabacum cv. Xanthi were loaded with the NO-specific fluorophore, diaminofluorescein, and subjected to an abiotic stressor. Light stress and mechanical injury had no apparent effect on NO production. In contrast, high temperatures, hyperosmotic stress, salinity and epi-illumination in a microscope all led to rapid surges in NO-induced fluorescence. The fluorescence originated from cells of the palisade mesophyll and across all epidermal cell types, including guard cells, subsidiary cells, and long and short trichomes. Fluorescence was evident first in the plastids, then in the nucleus and finally throughout the cytosol. Nicotiana plumbaginifolia cell suspensions expressing the calcium reporter aequorin provided evidence that, under hyperosmotic stress, NO participates in the elevation of free Ca2+ in the cytoplasm. The physiological significance of NO production in response to abiotic stressors is discussed.


Abbreviations
4-AF-DA

4-aminofluorescein diacetate

CLSM

confocal laser scanning microscope

carboxy-PTIO

2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide

DAF-2DA

4,5-diaminofluorescein diacetate

NBT

nitroblue tetrazolium

NO

nitric oxide

NOC-9

(Z)-1-N-methyl-N[6-(N-methylammoniohexyl) amino]diazen-1-ium-1,2-diolate

ROS

reactive oxygen species

SNP

sodium nitroprusside.

INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Nitric oxide (NO), a gaseous and highly unstable free radical, has been described both as a cytotoxin and as a cytoprotectant in plants (Leshem & Haramaty 1996; Beligni & Lamattina 1999a, 2001a). When applied at relatively high doses to plants, NO clearly perturbs normal metabolism. Exposure to NO reduced net photosynthesis in leaves of oats and alfalfa (Hill & Bennett 1970) and respiration in carrot cell suspensions (Zottini et al. 2002). NO concentrations above 10−6 m inhibited the expansion of leaf laminae, increased the viscosity of simulated thylakoid lipid monolayers, and potentially impaired photosynthetic electron transport in peas (Leshem et al. 1997; Leshem, Wills & Ku 1998). In haploid cultures of Taxus, high NO levels were associated with irreversible DNA fragmentation and cell death (Pedroso, Magalhaes & Durzan 2000). However, at lower, concentrations, NO promotes normal plant growth and development (Beligni & Lamattina 2001b). NO has been shown in several species to stimulate germination, root elongation, leaf expansion and phytoalexin production, to inhibit etiolation, to retard the onset of senescence and to induce stomatal closure (Noritake, Kawakita & Doke 1996; Gouvêa et al. 1997; Leshem et al. 1998; Beligni & Lamattina 2000; García-Mata & Lamattina 2001).

Exposures to low levels of NO have also been reported to improve the performance of plants under various types of environmental stress. Tolerance to drought, for example, is enhanced in wheat seedlings that have first been loaded with the NO-donor, sodium nitroprusside (SNP) (García-Mata & Lamattina 2001, 2002). Similarly, SNP-treated potato plants are more resistant both to methylviologen herbicides and to infection by Phytophthora infestans than are untreated plants (Beligni & Lamattina 1999b), and NO treatment of tobacco and soybean suspension cells triggers the expression of several defense-related genes, indicating that this radical plays a prominent role in defence against microbial pathogens (Delledone et al. 1998; Durner, Wendehenne & Klessig 1998). Tolerance to UV-B radiation has also been implicated; treatment of Arabidopsis thaliana with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), a scavenger of NO, prevented the up-regulation of transcription of the gene encoding chalcone synthase, a key enzyme in the flavonoid pathway that is believed to be important in conferring UV-B protection (Mackerness et al. 2001). NO is evidently highly versatile in its physiological effects.

Many plants are believed to produce substantial amounts of NO in their natural environments (Wildt et al. 1997). The possibility exists therefore that NO production occurs naturally as a generalized stress response (Magalhaes, Pedroso & Durzan 1999). Along with other compounds, such as abscisic acid, the jasmonates and ethylene, NO may function to mitigate the effects of disparate stressors in diverse plant taxa (Leshem & Kuiper 1996). Two interrelated mechanisms by which NO might abate stress have been proposed. First, NO might function as an antioxidant, directly scavenging the reactive oxygen species (ROS) that are generated by most stressors. NO can react rapidly with superoxide radicals to form peroxynitrite (Radi et al. 1991). Although peroxynitrite and its protonated form ONOOH are themselves oxidizing agents, they are considered to be less toxic than peroxides and may therefore limit cell damage (Wink et al. 1993). NO has been shown to significantly decrease superoxide formation in microsome suspensions (Caro & Puntarulo 1998). Second, NO may function as a signalling molecule in the cascade of events leading to gene expression (Wendehenne et al. 2001). These events involve both the activation of cGMP/Ca2+-dependent pathways and interactions with ROS (Durner et al. 1998; Klessig et al. 2000; Delledonne et al. 2001). The chemical properties of NO (small molecule, short half-life, absence of charge and high diffusivity) suggest that it would be an ideal inter- and intramolecular signalling molecule in plant stress responses (Foissner et al. 2000).

Most previous work on NO effects on stress responses in plants has involved the use of chemical donors, scavengers, or inhibitors of NO synthesis. Far fewer studies have recorded the natural rates of production of NO under stressful environments. Such studies are often difficult to compare because of the different methods used both to apply the stress and to measure NO generation. Nonetheless, the available data provide a good indication that NO biosynthesis in plants is responsive to disparate environmental cues: a 2 h heat stress exposure at 37 °C resulted in a doubling of the rate of NO emission from alfalfa sprouts (Leshem et al. 1998); leaves of Arabidopsis thaliana, and haploid cell suspensions of Taxus that were subjected to mechanical stress by centrifugation responded with eight- and five-fold increases in NO production, respectively (Pedroso et al. 2000; Garcês, Durzan & Pedroso 2001); and the combined stresses of mechanical injury (severing from the roots) and drought increased NO generation from pea shoots approximately 1.5 times (Leshem & Haramaty 1996). Biotic stressors also stimulate NO evolution. Cryptogein, a fungal elicitor from Phytophthora cryptogea, increased NO production four-fold in tobacco leaves (Foissner et al. 2000). In that study, NO was shown to be present in the plastids, nuclei, plasma membrane and possibly the peroxisomes of leaf epidermal cells.

Collectively, these experiments indicate that it is worth pursuing the hypothesis that NO emission occurs as a generalized stress response in plants. Here, we report the impacts of light, high temperatures, osmotic shock, salinity and mechanical injury on NO evolution from tobacco leaf cells, as measured both microscopically and fluorometrically. We present evidence for novel cellular sites of NO production. Finally, because abiotic stresses commonly lead to elevated Ca2+ levels in plant cells (Sanders, Brownlee & Harper 1999), we explore the possibility that stress-induced NO potentiates Ca2+ mobilization. Our data indicate that rapid and significant surges in NO can be induced by heat, osmotic and salinity stresses, but not by light stress or mechanical injury. NO appears to be involved in the mobilization of cytosolic free Ca2+ in response to hyperosmotic stress.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Plant material

Tobacco plants (Nicotiana tabacum cv. Xanthi) were grown from seed in pots under natural light in a greenhouse maintained at 25 ± 2 °C. Fully expanded leaf laminae were harvested immediately prior to the microscopy treatments. Fluorometric measurements were obtained using cell suspensions derived originally from the N. tabacum leaf mesophyll. The cells were cultured in Chandler's medium (Chandler, Tandeau de Marsac & Kouchkovsky 1972) on a rotary shaker (150 r.p.m) at 25 °C, with a constant irradiance of 15 µmol m−2 s−1 white light provided by Cool White fluorescent tubes (Osram, Munich, Germany). The cells were maintained in an exponential growth phase, and were subcultured 1 d before use. Nicotiana plumbaginifolia cells expressing the apoaequorin gene were cultured in Chandler's medium and maintained in dark-grown suspension by continuous shaking.

Microscopy

Peels containing the adaxial epidermis and uppermost palisade mesophyll were taken from N. tabacum leaf laminae under subdued lighting. They were incubated in the dark for 30 min at 25 °C in 10 mm Tris-HCl (pH 7.0) containing 10 µm 4,5-diaminoflurescein diacetate (DAF-2DA), the probe used for visualization of NO. The peels were washed for 10 min in fresh buffer, mounted in buffer on microscope slides, and then examined immediately under epifluorescence in a Leica DMRB microscope (Leica, Wetzlar, Germany) fitted with an Osram HBO 50 W short arc mercury lamp and an FITC filterset (excitation 490 nm; beamsplitter 510 nm; emission 525 nm). Additional peels were examined in a Leica TCS4D confocal laser scanning microscope (CLSM), excited with the 488 nm line of a krypton-argon laser, and emissions collected at 505–530 nm (probe-derived fluorescence) and ≥ 590 nm (chlorophyll autofluorescence). The production of green fluorescence under these conditions was attributed to the presence of NO (Foissner et al. 2000). To test the efficiency of the staining technique, some peels were incubated in 200 µm (Z)-1-N-methyl-N[6-(N-methylammoniohexyl) amino]diazen-1-ium-1,2-diolate (NOC-9), an efficient donor of NO to plant cells (Foissner et al. 2000), in addition to the buffered DAF-2 DA. To serve as negative controls, peels were incubated in 10 mm Tris-HCl alone.

After preliminary inspection, each mounted peel was subjected to one of the following treatments: (1) light: peels were epi-illuminated for various durations with monochromatic blue (450 nm) or green (490 nm) light, or else were given transmitted white light, as provided by the microscope; (2) temperature: microscope slides bearing the mounted peels were placed on a hot plate at 40 °C for various durations in the dark and then transferred to room temperature (approx. 25 °C); (3) mechanical injury: peels were pierced with a needle, or scored with a scalpel; and (4) osmotica: peels were re-mounted in 250 mm sorbitol or 250 mm NaCl. The peels from all treatments were inspected for green fluorescence at regular intervals until 30 min after treatment. All slides were held in darkness between inspections. Treatments were replicated at least three times, each using a peel from a different leaf. Images were captured on Fujichrome (Odawara, Japan) 400 ASA colour slide film. The slides were scanned into a computer, and fluorescence intensities quantified as mean greyscale values using Adobe Photoshop 5.0 (Adobe Systems, San Jose, CA, USA).

Fluorometric measurements

Nicotiana tabacum cell cultures were washed twice by filtration in a suspension buffer containing 175 mm mannitol, 0.5 mm CaCl2.2H2O, 0.5 mm K2SO4 and 20 mm HEPES. They were re-suspended at 0.1 g fresh weight (FW) mL−1 in the buffer plus 20 µm DAF-2DA, incubated in the dark for 1 h on a rotary shaker at 24 °C and then rinsed in fresh buffer. For the light, temperature, salinity and osmotic stress treatments, 0.75 mL of the final suspension was pipetted into each 16-mm-diameter well of a Costar (Cambridge, MA, USA) 24-well plate and its fluorescence (excitation 485 nm; emission 510 nm) recorded in a Fluoroskan Ascent fluorometer (Labsystems, Helsinki, Finland). Unless otherwise stated, the plates were held at 25 °C and agitated inside the fluorometer (120 r.p.m., orbital diameter 6 mm) in darkness between measurements. For the mechanical stress experiments, 2 mL aliquots of the cell suspension were dispensed into glass cuvettes and fluorescence was measured at 25 °C using a Kontron (Zurich, Switzerland) SFM-25 spectrofluorometer. The cells were then subjected to an environmental stressor and their fluorescence monitored at regular intervals.

Light treatments

Plates were constantly irradiated with white light from a Philips (Eindhoven, Netherlands) 400 W SON-T PLUS high pressure sodium lamp. To test for effects of light quantity, the irradiance was varied to between 10 and 400 µmol m−2 s−1 by adjusting the distance between the lamp and cells. A glass tank containing water, positioned beneath the lamp, eliminated UV radiation and reduced heat build-up. To test for effects of light quality, individual wells were irradiated with 20 µmol m−2 s−1 white, red (660 nm) or green (510 nm) light supplied by the fibre optic arm of a Schott KL1500 150 W collimated halogen light source (Schott, Mainz, Germany) and passed through coloured cellulose filters (Supergel Rosco, Sydenham, UK).

Temperature treatments

The ambient temperature inside the fluorometer was raised from 25 to 45 °C over a 5-min period using the dedicated software controls and then held at 45 °C for the duration of the experiment.

Osmotic and salinity treatments

The cell suspensions were supplemented with either 250 mm sorbitol or 250 mm NaCl.

Mechanical stress

The cell suspension in each cuvette was stirred vigorously with an 8-mm-long stir bar on a magnetic stirrer set at approximately 300 r.p.m. To provide aeration, the magnetic stirrer was itself positioned on a rotary shaker, so that treated cells were both rotated and stirred. Control cells were aerated on the rotary shaker, but were not stirred. All cells were held in the dark between measurements. Cell integrity and viability 15 min after the treatment were assessed under a compound microscope upon staining with 1% (w/v) neutral red.

4-AF-DA and carboxy-PTIO treatments

To verify that increases in green epifluorescence were attributable to NO evolution, further suspensions were loaded for 1 h with 4-aminofluorescein diacetate (4-AF-DA) rather than DAF-2DA, washed and then subjected to abiotic stressors. As a second control, DAF-2DA loaded cells were incubated for 10 min in 500 µm carboxy-PTIO, a NO scavenger, prior to treatments.

Efficiency of carboxy-PTIO in NaCl treatments

To test for possible interference by NaCl with the capacity for carboxy-PTIO to scavenge NO, a solution of (1) NOC-9 (200 µm); (2) NOC-9 (200 µm) + carboxy-PTIO (500 µm); (3) NOC-9 (200 µm) + NaCl (250 mm); or (4) NOC-9 (200 µm) + carboxy PTIO (500 µm) + NaCl (250 mm) was added to cells loaded with DAF-2DA. Peak fluorescence levels were recorded 15 min after treatment.

Cytosolic Ca2+ concentrations

The transgenic N. plumbaginifolia cell suspensions were washed and re-suspended in buffer at a final concentration of 0.1 g FW mL−1. In vivo reconstitution of aeqorin was performed by the addition of 2 µL coelenterazine to 10 mL cell suspension for at least 3 h in the dark before subjecting the cells to an abiotic stressor. Bioluminescence was measured using a digital luminometer (Lumat LB9507; Berthold, Bad Wildbad, Germany) as described by Lecourieux et al. (2002). At the end of each experiment, residual functional aequorin was quantified by adding 300 µL lysis buffer (10 mm CaCl2, 2% Nonidet P-40 and 10% ethanol) and monitoring the resultant increase in luminescence. Luminescence data were converted to calcium concentrations using the equation established by Allen, Blinks & Prendergast (1977).

Chemicals

DAF-2DA and 4-AF-DA were purchased from Calbiochem (La Jolla, CA, USA) and carboxy-PTIO and NOC-9 were from Alexis Biochemicals (Lausen, Switzerland). All other chemicals were from Sigma (St. Louis, MO, USA).

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

When DAF-2DA is loaded into plant cells it is converted by cytosolic esterases to DAF-2, which reacts with N2O3, derived from the oxidation of NO, to form a green fluorescent triazole derivative, DAF-2T. The reaction is considered to be specific (Suzuki et al. 2002), and thus the appearance of green fluorescence in DAF-2DA-treated cells can serve as a useful diagnostic tool for locating and quantifying NO emission.

Imaging of NO in tobacco leaves challenged by abiotic stressors

Tobacco leaf peels that had been incubated in buffer, but not DAF-2DA, did not fluoresce green when observed by epifluorescence microscopy. Instead, most cells fluoresced red, attributable to the autofluorescence of chlorophyll, which is abundant across all epidermal cell types on both lamina surfaces. In contrast, peels that had been loaded with both DAF-2DA and the NO-donor, NOC-9, rapidly exhibited strong green fluorescence from the cell walls, cytoplasm and intercellular spaces of all cells (data not shown). These observations provided a useful starting point for our experiments, indicating both that the probe had penetrated all cells at sufficient concentrations to detect NO, and that measurements of green fluorescence would not be compromised by green autofluorescence from cell components.

Epi-illumination of DAF-2DA-load peels with blue (450 nm) or green (490 nm) light from the microscope resulted in a rapid evolution of NO. Green fluorescence was first detectable 1 min after light exposure, and increased linearly in intensity to a maximum after 7 min (Fig. 1). In contrast, levels of green fluorescence from the non-exposed regions remained low, and were restricted to isolated cells or sporadic cell clusters (Fig. 2). Maximum fluorescence levels (mean greyscale values attributable exclusively to green fluorescence) were on average 1.9 times greater in the illuminated than in the non-exposed regions. The exposure of peels to transmitted white light did not significantly increase green fluorescence beyond background values.

image

Figure 1. Fluorescence kinetics for DAF-2DA-infused peels from tobacco leaves in response to epi-illumination with green light. Fluorescence was quantified as mean greyscale values for captured images after the subtraction of values for chlorophyll autofluorescence. Data show means ± standard errors for four exposed regions of one peel photographed over time. The data are representative of three replicates using peels from different leaves. Fluorescence of non-exposed regions did not increase beyond the initial value.

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image

Figure 2–17. Fluorescence of DAF-2DA-infused tobacco leaf cells in response to stressors. Figure 2.  Peel taken from adaxial epidermis. Cells beneath the dotted line had been epi-illuminated with green light for 6 min; those above the line had been held in the dark. Figures 3 & 4. CLSM micrographs of stomatal guard cells 1 min (Fig. 3) and 7 min (Fig. 4) after epi-illumination with green light. Upper images show combined fluorescence from chlorophyll (red) and DAF-2T (green); lower images show same cells after subtraction of red fluorescence. Figure 5. CLSM micrograph of stomatal subsidiary cells after 5 min epi-illumination showing DAF-2T fluorescence in nuclei, plastids and cytosol. Figure 6. Adaxial epidermal cells located above the petiole (upper portion of image) and lamina (lower portion) after epi-illumination. Figure 7. CLSM micrograph of paradermal optical section through uppermost palisade mesophyll after epi-illumination of adaxial surface of leaf, before (upper) and after (lower) subtraction of chlorophyll autofluorescence. Figure 8. Stomatal guard cells responding to 250 mm sorbitol. Figure 9. Adaxial epidermal cells after 7 min heat treatment at 40 °C. Figures 10 & 11. Light micrograph (Fig. 10) and autofluorescence micrograph (Fig. 11) of long trichomes. Figure 12. CLSM micrograph of heat-treated long trichome, before (upper) and after (lower) subtraction of chlorophyll autofluorescence. Figure 13. DAF-2T fluorescence in various subapical positions in heat-treated long trichomes. Figures 14–16. Short trichomes after heat treatment as observed by light (Fig. 14) and epifluorescence (Figs 15 & 16) microscopy. Exudation of DAF-2T is evident (arrow). Figure 17. Fluorescence from epidermal cells along wounded edge of leaf. Bars: Figs 2, 6, 8, 9, 10, 11, 13,14–17 = 100 µm; Figs 3–5 = 10 µm; Figs 7 & 12 = 25 µm.

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NO evolution, as observed by green fluorescence associated with the epi-illumination of tobacco leaf peels, was detected in all epidermal cell types (Figs 3–6). Guard cells were normally the first cell type to respond; fluorescence was evident first in the plastids (Fig. 3), then in the nucleus (Fig. 4), and eventually throughout the cytosol. As the epi-illumination treatment progressed, similar patterns of fluorescence development were observed in the stomatal subsidiary cells (Fig. 5), in epidermal cells located above both lamina and midrib (Fig. 6), and in the uppermost layer of palisade mesophyll (Fig. 7). However, departures in the pattern were noted; some cells, for example, developed fluorescence in the nucleus but not in the chloroplasts. Approximately 20% of the epidermal cells of all types did not respond to the treatment.

Responses of peels to the sorbitol, salinity and heat treatments were similar to one another. All promoted a rapid surge of fluorescence, especially from the stomatal guard cells (Fig. 8), the epidermal cells located above the midrib and major veins (Fig. 9), and trichomes (Figs 10–16). Responses were detectable within 1 min of treatment, but fluorescence was most intense after 10 min. Heat treatments as short as 20 s at 40 °C were sufficient to elicit a response, although more cells fluoresced with longer treatments up to 7 min.

Tobacco leaves have two morphologically distinct types of trichomes, termed ‘long’ and ‘short’ (Meyberg et al. 1991; Choi et al. 2001). The long trichomes consist of a uniseriate ‘stalk’ up to five cells long, and a glandular ‘head’ comprising two or three cells (Fig. 10). Long trichomes were found throughout both surfaces of tobacco leaves, but they were particularly abundant on the petiole and above major veins. All of the cells in the long trichomes were chlorophyllous; they autofluoresced red upon excitation with blue light, both in the control and the DAF-2DA-loaded peels (Fig. 11). Upon heat, salinity and sorbitol treatments of the loaded peels, however, a proportion (approximately 20%) of the long trichomes, particularly those located above veins or adjacent to the wounded edges, fluoresced intensely green. Fluorescence originated from the chloroplasts, and was normally strongest in the apical cell of the glandular head (Fig. 12), although subapical locations, and various combinations of stalk and head cells were also noted (Fig. 13). Both the frequency of fluorescing trichomes and the intensity of their fluorescence in-creased appreciably in the heat-treated peels under epi-illumination during the microscopic observation.

Short trichomes, in contrast, have a stalk comprising only one or two cells and a disproportionately large, multicellular glandular head (Fig. 14). They are most abundant on the lamina surfaces between veins. Fluorescence of these trichomes in heat-, saline- and sorbitol-treated peels was less obvious than that for the long trichomes. The cell walls of short trichomes in the control peels (without DAF-2DA) emitted a pale yellow autofluorescence; this may have partially masked green fluorescence from the loaded peels. Nonetheless, strands of green fluorescence demarcating cell wall boundaries were detectable in the glandular heads after prolonged heat treatment (Fig. 15). Perhaps significantly, 20 min after a 7-min heat treatment many short trichomes were observed exuding droplets of the fluorescent triazole derivative from the glandular head into the surrounding buffer (Fig. 16).

Mechanical wounding of DAF-2DA-loaded peels did not produce consistent results. Some peels responded rapidly to injury, evolving intense fluorescence predominantly from stomata and long trichomes, but most did not respond, or else emitted fluorescence only gradually over the 30 min of observation. All DAF-2DA-loaded peels, however, fluoresced intensely at their cut edges (Fig. 17).

Fluorometric measurements of NO in cell suspensions

None of the light treatments significantly increased fluorescence output from the cell suspensions relative to background levels (P > 0.05; Fig. 18a & b). White, red (660 nm) and green (510 nm) light were equally ineffective at all tested irradiances. On the contrary, fluorescence levels diminished slightly when cells were irradiated with 400 µmol m−2 s−1 white light (Fig. 18b), although the decline was not statistically significant (P > 0.05).

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Figure 18. Nitric oxide production by tobacco cell suspensions during light treatments. (a) Cells at 25 °C irradiated with 20 µmol m−2 s−1 white light (○), and dark controls (•). Data show mean fluorescence values (n = 10) ± SE. (b) Cells were held at 25 °C in the dark for 30 min, then irradiated for 30 min with 150 µmol m−2 s−1 white light, and then for 30 min with 400 µmol m−2 s−1 white light. Data are means (n = 8) ± SE

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Fluorescence attributable to DAF-2T was significantly greater from those cells held at the higher temperatures. When cell temperatures were raised from 25 to 45 °C using the fluorometer controls, fluorescence levels increased by 80% within 5 min (Fig. 19). The increase was reproducible and highly significant (P < 0.0001). In contrast, no increase in fluorescence was observed when cells were loaded with 4-AF-DA instead of DAF-2DA (Fig. 19). Lacking an amino group that constitutes the NO-reactive site in DAF-2DA, 4-AF-DA does not react with NO to form a fluorescent product; its use therefore confirmed that the fluorescence surge was not attributable to autoflorescence of components leaking from thermally injured cells, or from degradation products of the probe. Carboxy-PTIO delayed and significantly reduced the extent of heat-induced DAF-2T fluorescence over the first 60 min of treatment, although its effect was transient (Fig. 19).

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Figure 19. Nitric oxide production by tobacco cell suspensions in response to heat treatments. Cell temperatures were increased from 25 to 45 °C over a 5 min period from the point marked with an arrow, and then held at 45 °C. Control cells (•); cells loaded with 4-AF-DA (□); cells loaded with DAF-2DA in the absence (○) or presence (▵) of 500 µm carboxy-PTIO. Data are means (n = 8) ± SE

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The responses of cells to sorbitol were rapid and pronounced. All of the sorbitol replicates gave a significant surge in fluorescence within the first 2 min of treatment, followed by a partial decline (Fig. 20a). Fluorescence values from sorbitol-treated cells were three-fold greater than those from untreated cells after 2 min (P < 0.001) and remained 35% greater after 30 min (P < 0.001). Carboxy-PTIO effectively reduced the fluorescence of sorbitol-treated cells to background levels (Fig. 20a).

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Figure 20. Nitric oxide production by tobacco cell suspensions in response to sorbitol and salinity. (a) Control cells with DAF-2DA (•), 250 mm sorbitol (○), 250 mm sorbitol plus 500 µm carboxy-PTIO (▵). (b) Control cells with DAF-2DA (•), cells loaded with 4-AF DA and incubated with 250 mm NaCl (□), DAF-2DA loaded cells incubated with 250 mm NaCl in the absence (○) or presence (▵) of 500 µm carboxy-PTIO. (c) Ability of carboxy-PTIO to scavenge NO released by the NO donor NOC-9 in cultured tobacco cells in the absence or presence of 250 mm NaCl. Data are means (n = 12) ± SE

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The fluorescence from NaCl-treated cells (Fig. 20b) increased progressively to a value 80% greater than that of untreated cells after 30 min (P < 0.0001). In some of the replicates, this increase was preceded by a transient spike. In contrast to the sorbitol treatment, however, carboxy-PTIO did not mitigate the surge in NaCl-mediated fluorescence (Fig. 20b). Cells that had been infused with 4-AF-DA instead of DAF-2DA did not fluoresce upon NaCl treatment, indicating that the surge was not an artefact of the probe under these conditions. To understand the absence of effects of carboxy-PTIO on the NaCl-induced fluorescence, the capacity for carboxy-PTIO to scavenge NO released by the donor, NOC-9, was measured in cultured tobacco cells in the absence or presence of 250 mm NaCl. In the absence of NaCl, carboxy-PTIO eliminated up to 95% of the fluorescence originating from NOC-9 (Fig. 20c). However, when NaCl was present, the scavenging efficiency of carboxy-PTIO was reduced by 40% (Fig. 20c).

Cells that had been dispensed into glass cuvettes for the mechanical stress experiment generated higher background fluorescence counts than did those in the wells, reflecting the greater number of cells used. The imposition of mechanical stress by stirring the cells with a magnetic stir-bar increased fluorescence output further (Fig. 21). Fluorescence increased approximately linearly over time, culminating in a 60% increase over the 20 min observation period (P < 0.0001). However, similar increases in fluorescence were observed both when 4-AF-DA was substituted for DAF-2DA and, to a lower degree, in the absence of either compound. Moreover, the scavenger carboxy-PTIO did not significantly diminish the fluorescence elevation (Fig. 21). Collectively, the data indicated that the increase of fluorescence under mechanical stress was not attributable to NO.

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Figure 21. Nitric oxide production by tobacco cell suspensions in response to mechanical stress. Cells were continuously stirred during the experiment. Control cells minus DAF-2DA (•), cells loaded with 4-AF DA (▪), DAF-2DA loaded cells in the absence (○) or presence (▵) of 500 µm carboxy-PTIO. Data are means (n = 4) ± SE

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NO participation in cytosolic Ca2+ mobilization

All of the abiotic stresses used in this study have been previously shown to induce a rapid and transient increase in free cytosolic Ca2+ concentration ([Ca2+]cyt) (Reddy 2001). Studies with animal cells indicate that NO activates Ca2+ channels, leading to elevated [Ca2+]cyt which in turn stimulate diverse physiological processes (Willmott et al. 1996). To test that NO plays a similar role in plants in response to abiotic stress, we looked for effects of NO-scavenging (by carboxy-PTIO) on aequorin-transformed cell suspensions of Nicotiana plumbaginifolia. These transgenic cells constitutively expressed the Ca2+ reporter aequorin in the cytosol, the luminescence of which was proportional to [Ca2+]cyt. As the efficacy of carboxy-PTIO as an NO-scavenger was apparently impaired by salinity and heat, we focused our examination on the responses to hyperosmotic stress induced by sorbitol treatment.

In the absence of carboxy-PTIO, sorbitol led to two successive [Ca2+]cyt elevations: the first, a transient spike reaching 1.2 µm, occurred within 20 s, and the second, a more sustained increase, peaked at 0.6 µm after 17 min (Fig. 22). In the presence of carboxy-PTIO, the heights of both elevations were reduced by approximately 35%, and the second [Ca2+]cyt peak was delayed. Control cells (lacking both sorbitol and carboxy-PTIO, or with cPTIO alone) consistently yielded [Ca2+]cyt values of around 120 nm throughout the experiment (data not shown). These results indicate that in response to sorbitol, NO participates in the elevation of [Ca2+]cyt.

image

Figure 22. Effect of carboxy-PTIO on [Ca2+]cyt changes in response to 0.25 m sorbitol treatment in aequorin-transformed cells of Nicotiana plumbaginifolia. Sorbitol was added to the cell suspensions at time 10 min (point marked with an arrow). Data show one experiment, representative of six replicates.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Environmental stressors lead to the production of free radicals in plants. Species of reactive oxygen have been shown to increase appreciably in response to stressors as diverse as high light, drought, mechanical injury, temperature extremes, heavy metals, salinity, UV radiation, ozone, herbicide treatments and pathogens (reviewed by Foyer, Lelandais & Kunert 1994; Alscher, Donahue & Cramer 1997). Far less is known of the potential role of reactive nitrogen in plant stress responses. Here, we considered the possibility that levels of NO, one of the key reactive nitrogen species, might also respond to diverse environmental cues. Using DAF-2 for the detection and imaging of NO, the leaf peels and cell suspensions provided a tractable and reproducible system to locate and quantify NO emission. Clearly, both the peels and the cell suspensions are themselves stressed biological systems, as evidenced by the background fluorescence counts from control cells. Therefore, only the difference in magnitude of fluorescence between control and treated cells represented the response to an abiotic stressor. Our data indicate that tobacco cells, when challenged by high temperatures, osmotic stress, or salinity, generate a rapid and significant surge in NO levels. These same cells do not, however, yield NO in response to light of up to 400 µmol m−2 s−1 and to mechanical stress imposed by wounding or cell agitation (Figs 18 & 21). Thus, although the synthesis of NO can be triggered by several, disparate stressors, it cannot be considered a universal plant stress response.

Our analysis assumes that DAF-2, a fluorophore widely used as a diagnostic for NO, is indeed selective for NO and the products of its oxidation. The specificity of DAF-2 has been rigorously tested. Joud’heuil (2002) concluded that the products derived from the autoxidation of NO are essential for the nitrosation of DAF-2 to the fluorescent form DAF-2T, although the probe can under certain conditions react both with N2O3 (from the oxidation of NO) and with the NO formed from the dissociation of peroxynitrite. Thus, measurements of DAF-2T fluorescence can, if anything, overestimate rates of NO evolution. This caveat does not compromise our own experiments for which only total amounts of NO were required, both from direct and indirect sources. Of greater concern was a recent report of possible interference in NO measurements from the fluorescence generated by the reactions of DAF-2 with ascorbate and dehydroascorbate (Zhang et al. 2002). Such interference might well have contributed to the background fluorescence of our tobacco cells after 1-h incubation in DAF-2DA. The kinetics of the reactions with ascorbate and dehydroascorbate, however, are slower than could account for the rapid surges in fluorescence observed in our stress experiments. Nonetheless, we attempted to verify that the green fluorescence was indeed attributable to DAF-2T, rather than to the autofluorescence of other products released from the cells during the stress treatment, or from their reactions with the probe. When tobacco cells suspensions were incubated in the probe homologue 4-AF-DA, which does not form DAF-2T, none of the abiotic stressors other than mechanical injury triggered a significant increase in fluorescence. The fluorescence from DAF-2DA-infused cells under heat, sorbitol and NaCl treatments could be reasonably ascribed therefore to the formation of DAF-2T.

To assess DAF-2DA specificity further, we examined the effects of the NO scavenger, carboxy-PTIO, on fluorescence levels. Carboxy-PTIO should remove all NO before it reacts with DAF-2, thereby abolishing the green fluorescence. Unexpectedly, the effects of carboxy-PTIO varied according to the nature of the stress applied. Carboxy-PTIO completely suppressed the sorbitol-mediated fluorescence (Fig. 20a), its effect on heat-mediated fluorescence was transient (Fig. 19) and the NaCl and mechanical stress treatments were insensitive to this scavenger (Figs 20b & 21). Certain abiotic stressors apparently compromise the ability of carboxy-PTIO to scavenge NO. Consistent with this hypothesis, we observed that in the presence of 250 mm NaCl, carboxy-PTIO was less effective in reducing DAF-2T fluorescence induced by the NO donor, NOC-9 (Fig. 20c). The degree to which NaCl affected carboxy-PTIO activity was smaller in NOC-9 treated cells (approximately 40% inactivation) than in cells treated with NaCl alone (almost 100%). This probably indicates that the kinetics of carboxy-PTIO inactivation are relatively slow. NO was delivered rapidly by NOC-9, within seconds as observed under a microscope. NO is probably released from NOC-9 before the biochemical events that impair carboxy-PTIO activity (for example, by modification of cellular pH or of the redox balance that causes reduction or oxidation of carboxy-PTIO) are potentiated, presenting sufficient opportunity for the scavenging of approximately 60% NO before carboxy-PTIO is inactivated. In contrast, the natural production of NO by cells in response to NaCl began only 5 min after treatment and was maximal after 30 min (Fig. 20b), by which time carboxy-PTIO would have been fully inactivated. Supportive of this hypothesis, we observed that the capacity for carboxy-PTIO to scavenge NO released by NOC-9 declined significantly with the duration of exposure to NaCl (data not shown). We recommend caution in interpretations based on the use of carboxy-PTIO in plant stress experiments.

Mechanical injury appears unlikely to cause the production of NO in tobacco leaves. The increase in green fluorescence, which was noted for DAF-2DA-loaded cells upon vigorous stirring, also occurred in control cells that either contained no probe or else were loaded with the non-fluorescent analogue, 4AF-DA. Moreover, the fluorescence was not abolished by carboxy-PTIO. Microscopic observations of DAF-2DA-loaded peels consistently showed fluorescence at their cut edges (Fig. 17); at those locations, however, cells would also be most prone to the effects of desiccation and/or osmotic imbalance. It is probable therefore that mechanical stress triggers the release of cellular constituents, or else the generation of new compounds, that have fluorescence emission profiles similar to that of DAF-2T. Our conclusion is consistent with that of Orozco-Cardenas & Ryan (2002), who found no difference in NO production between leaflets from wounded and control tomato plants. Tobacco and tomato apparently differ from Taxus, for which NO levels increased significantly when the cells were centrifuged (Pedroso et al. 2000).

The non-inducibility of NO by light is surprising given that (1) the cells had acclimated to low irradiance (15 µmol m−2 s−1) prior to treatment and are therefore likely to have been strongly photo-inhibited during the high light treatments; (2) the epi-illumination of leaf peels in a microscope led to a rapid burst of NO synthesis (Figs 1 & 2); and (3) a previous study using intact plants from various species reported NO emissions as high as 1.5 × 10−14 mol cm−2 s−1 under strong light (Wildt et al. 1997). However, the short arc mercury lamp used for epi-illumination in our experiment generated considerable heat in addition to the light. We recorded up to 15.9 °C increases in temperature of the microscope slide after only 30 s epi-illumination (data not shown). The epi-illumination effect is almost certainly a response to the heat rather than to light, since transmitted white light from the microscope (from a tungsten-halogen lamp with negligible heat output) failed to elicit green fluorescence in DAF-2-infused peels. Moreover, light has been reported to suppress NO synthesis in sunflower leaves and green alga cell cultures, which yield substantial surges in NO when they are returned to darkness (Mallick et al. 2000; Rockel et al. 2002; Sakihama, Nakamura & Yamasaki 2002). Those authors have suggested that by promoting nitrite reductase activity, light decreases the pool of cytoplasmic nitrite which might otherwise be converted to NO through nitrate reductase. Consistent with their results, we found that NO production was slightly reduced when tobacco cell suspensions were irradiated with strong light (Fig. 18b). Collectively, our experiments demonstrate that although light per se does not stimulate NO production in tobacco leaves, the evolution of NO is possible under light when the cells are challenged with an additional stressor, such as heat.

The cellular sites of NO synthesis are more diverse than has been documented previously. All epidermal cell types – guard cells, subsidiary cells, trichomes, costal and intercostal epidermal cells – as well as the palisade mesophyll cells apparently have the potential to generate NO (Figs 2–17). In agreement with previous studies (reviewed by Wendehenne et al. 2001), NO production was primarily associated with the chloroplasts, although subsequent nuclear (Fig. 4) and general cytosolic (Fig. 9) locations were also noted. It is probably significant that the guard cells and the trichomes were normally the first cell types to respond to stress, since these are obvious conduits for the rapid emission of NO into the atmosphere. Tobacco leaf trichomes have been shown previously to secrete diterpenes, sucrose esters, various alkaloids and even cadmium crystals (Zador & Jones 1986; Lin & Wagner 1994; Choi et al. 2001). Our observations of DAF-2T fluorescence in various cells of the long trichome stalk (Fig. 13) and of the exudation of DAF-2T from the heads of short trichomes (Fig. 16) are consistent with the hypothesis that trichomes also secrete NO and its derivatives.

Although the mechanism(s) through which NO impacts on plant stress responses remains unresolved, our observation that NO participates in the biphasic increase in [Ca2+]cyt triggered by sorbitol indicates that it is involved in calcium mobilization. It is intriguing that the first elevation of cytosolic Ca2+ occurred almost immediately upon sorbitol treatment (Fig. 22), yet NO production peaked 2 min later (Fig. 20a). Given that carboxy-PTIO altered the profile of the first Ca2+ spike less than that of the second spike, we postulate that this first increase in cytosolic Ca2+ is largely NO-independent and, indeed, may itself activate the NO-generating enzyme. In turn, once produced, NO may contribute to the second Ca2+ elevation by activating Ca2+ channels. Similar observations have been reported for animal cells (Willmott et al. 1996) and are consistent with studies in which Ca2+ was shown to mediate NO-induced accumulation of defence genes in tobacco (Durner et al. 1998; Klessig et al. 2000). Carboxy-PTIO did not modify the [Ca2+]cyt signatures formed in response to heat or NaCl (data not shown). However, we cannot rule out the possibility of Ca2+ mobilization by NO under such conditions because carboxy-PTIO apparently performs poorly under these stressors. Further experimentation is required to establish the potential link between NO and the second messenger Ca2+ in the signalling cascade activated by abiotic stress.

The emission of NO from leaves constitutes a small deficit to the nitrogen economy of a plant. Clearly, unnecessary losses in nitrogen would not per se be advantageous to a plant under stress. It is of value therefore to consider whether NO occurs as the default product of a perturbed metabolism, serving no purpose, or instead is useful in abiotic stress tolerance. Recently, NO has been shown to mediate the ABA-induced closure of stomata in Pisum and Arabidopsis (Neill et al. 2002). NO may have a pivotal role therefore as a signalling molecule in the protection from the effects of drought. The emission of NO via stomata (Figs 3 & 4) and/or trichomes (Figs 12–16) would also directly moderate the overall oxidative load. Unlike the oxygen radicals, NO is highly diffusible; this could be especially beneficial when stressors restrict or block photosynthetic electron transport. Under such conditions, NAD(P)H converts cytosolic nitrite to NO by nitrate reductase (Yamasaki 2000; Sakihama et al. 2002) and the NO would diffuse rapidly into the atmosphere. The generation and emission of NO could therefore be an effective way to dissipate excess free radicals, supplementing other detoxification mechanisms. However, possible advantages of NO evolution have yet to be demonstrated in the field.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

We thank C. Humbert for his assistance with the confocal laser scanning microscopy, and M.R. Knight and A.J. Trevawas for donating the N. plumbaginifolia cell line. The work was funded by a University of Auckland Research and Study Leave grant to K.S.G., by the Institut National de la Recherche Agronomique (Département Santé des Plantes et Environnement, Grant 1088-01a), the Ministère de l’Education Nationale de la Recherche et de la Technologie, and the Conseil Régional de Bourgogne.

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  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
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