Nitric oxide production by the differentiating xylem of Zinnia elegans

Authors


Author for correspondence: A. Ros Barceló Tel: +34 968364945 Fax: +34 968363963 Email: rosbarce@um.es

Summary

  • • Nitric oxide (NO) is currently regarded as a signal molecule involved in plant cell differentiation and programmed cell death.
  • • Here, we investigated NO production in the differentiating xylem of Zinnia elegans by confocal laser scanning microscopy to answer the question of whether NO is produced during xylem differentiation.
  • • Results showed that NO production was mainly located in both phloem and xylem regardless of the cell differentiation status. However, there was evidence for a spatial NO gradient inversely related to the degree of xylem differentiation and a protoplastic NO burst was associated with the single cell layer of pro-differentiating thin-walled xylem cells. Confirmation of these results was obtained using trans-differentiating Z. elegans mesophyll cells. In this system, the scavenging of NO by means of 2-phenyl-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide (PTIO) inhibits tracheary element differentiation but increases cell viability.
  • • These results suggest that plant cells, which are just predetermined to irreversibly trans-differentiate in xylem elements, show a burst in NO production, this burst being sustained as long as secondary cell wall synthesis and cell autolysis are in progress.

Introduction

The xylem constitutes the longest pathway of water transport in vascular plants. It is a simple pathway of low resistance, which enables water transport in large quantities with great efficacy, especially from the roots to the leaves. Most terminally differentiated cells fulfil specialized functions until they die but, in the case of the xylem, its function does not begin until after cell death. Thus, maturation of xylem elements involves cell death, so that functional water-conducting cells have no membranes or organelles, and what remains are the thick, lignified cell walls, which form hollow tubes through which water can flow with relatively little resistance (Kozela & Regan, 2003). Terminal xylem elements (water-conducting cells) are thus internally coated with lignins, which are three-dimensional phenolic heteropolymers covalently associated to the polysaccharide matrix of xylem cell walls (Anterola & Lewis, 2002). Lignins are also localized in other supporting tissues, such as the phloem fibres, and result from the oxidative polymerization of three monolignols (p-coumaryl, coniferyl and sinapyl alcohols) in a reaction that can be mediated by both laccases and peroxidases (Ros Barceló, 1997), leading to an optically inactive hydrophobic heteropolymer (Ralph et al., 1999). The process of sealing plant cell walls through lignin deposition is known as lignification and provides mechanical strength to the stems, protecting cellulose fibres from chemical and biological degradation (Grabber et al., 1988). In this context, plant cell wall lignification is one of the main restrictive factors in the use and recycling of plant biomass (Anterola & Lewis, 2002).

Programmed cell death is one of the most distinctive characteristics exhibited by the differentiating xylem in plants (Fukuda, 1996; Robert & McCann, 2000) and is invariably coordinated with processes of secondary cell wall formation and lignification (Groover & Jones, 1999). Execution of cell death in xylem elements involves a Ca2+ influx into the cell, and it is manifested by a rapid collapse of the vacuole leading to the release of hydrolytic enzymes, and the cessation of cytoplasmic streaming (Groover & Jones, 1999). To coordinate cell wall synthesis and cell death, a serine protease is secreted, while specific proteolysis of the extracellular matrix is necessary and sufficient to coordinate both processes (Groover & Jones, 1999). Although the metabolic cascade leading to cell death in xylem cells is partly known, little is known about the diffusible cell signal that switches this unavoidable process off. One of the possible signals is nitric oxide (NO), since NO is currently regarded as a signal molecule involved in plant cell differentiation and programmed cell death (Wendehenne et al., 2001; Neil et al., 2003). However, to our knowledge, there are no available data confirming that differentiating xylem cells are capable of synthesizing NO, and so both the nature and the extension of NO production by the differentiating xylem remains unknown. This is especially important since NO appears to be a key upstream signal of the last step in lignin biosynthesis, i.e. the monolignol assembly (Ferrer & Ros Barceló, 1999; Ros Barcelóet al., 2002a).

In this report, we studied NO production in the differentiating xylem of Zinnia elegans vascular bundles by means of confocal laser scanning microscopy (CLSM) using 4,5-diaminofluorescein diacetate (DAF-DA) as fluorescent probe (Foissner et al., 2000). Studies on NO production were extended to trans-differentiating Z. elegans mesophyll cells. In both cases, results showed that differentiating (noncollapsed) thin-walled xylem cells showed a burst of NO production just before the late processes of secondary cell wall formation and cell autolysis, making this the first report that suggests a certain role for NO in xylem differentiation/cell death.

Materials and Methods

Chemicals

Aminoguanidine, DAF-DA, Griess reagent, haemoglobin, Nw-nitro-d-arginine methyl ester (D-NAME), Nw-nitro-l-arginine methyl ester (L-NAME), NO, 2-phenyl-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide (PTIO) and sodium azide were purchased from Sigma-Aldrich (Madrid, Spain). The remaining chemicals used were obtained from various suppliers and were of the highest purity available.

Plant material

Seeds of Z. elegans L. cv. Envy were grown in a glasshouse under daylight conditions at 25°C as described by Ros Barceló (1998). The first internode (stem) 3 d and 6 d after the appearance of the first pair of true leaves was used for these studies. In the case of Z. elegans trans-differentiating mesophyll cell cultures, the first true leaves of 14-d-old seedlings were removed, surface-sterilized and rinsed in sterile distilled water. Single cells were isolated and cultured for 6 d in a differentiating medium, as described elsewhere (Fukuda & Komamine, 1982; López-Serrano et al., 2004).

Determination of lignins

Zinnia elegans stem cell walls were prepared by a Triton X-100 washing procedure (Ros Barcelóet al., 2002b). Total lignin content was determined by the acetyl bromide/acetic acid method exactly as described by Ros Barcelóet al. (2002b). Lignins were also detected using the Wiesner test by soaking 500-µm thick sections in the phloroglucinol reagent, which was composed of 1% (w : v) phloroglucinol in 10.1 m HCl/ethanol (25 : 75, v : v) (Pomar et al., 2002).

Polarized light microscopy

Stem sections were thoroughly washed with 50 mm Tris-maleate buffer, pH 7.5, fixed in 3% glutaraldehyde at room temperature and postfixed with 1% OsO4. Following postfixation, samples were washed, dehydrated and embedded as described by Ros Barcelóet al. (1991). Serial, thin sections (1–3 µm) were simultaneously observed by light microscope either after staining with toluidine blue (TB) (Ros Barcelóet al., 1991) or without TB staining using polarized light. Light micrographs were taken with a Leica DMRB microscope (Leica Mikrosysteme GmbH, Bensheim, Germany).

Confocal laser microscopy

Nitric oxide production was monitored by CLSM (Foissner et al., 2000). For this, either 500-µm thick stem sections, or trans-differentiating tracheary elements, were incubated in a loading buffer containing 10 mm Tris-HCl, pH 7.2, and 10 µm DAF-DA for 45 min, and then washed three times with the loading buffer without DAF-DA to remove the excess of fluorophore. Measurement of the absorbance at 260 nm of the washing solutions showed that this three-step washing protocol removed about 98.5% of the dye. Fluorescence images were visualized using a Leica TCS SP2 confocal laser microscope. Plant samples were excited with the 488 line of an argon laser and dye emissions were recorded using a 500–535 nm bandpass filter. Controls were performed in the presence of 100 µm PTIO. Fluorescence images were monitored in 25 sections selected at random from 20 to 30 plants, with 300 vascular bundles being observed in each assay. To study the effect of inhibitors, samples were preincubated for 30 min in L-NAME (1.0 mm), D-NAME (1.0 mm), aminoguanidine (1.0 mm) or sodium azide (100 µm). The images acquired were processed using the Microsoft Photo Editor and relative pixel intensities determined using the LCM lite (Leica) software.

To assess the effect of inhibitors on the quenching of the triazolofluorescein green fluorescence (TFGF), Z. elegans seedlings were homogenized in 50 mm Tris-HCl buffer, pH 7.2. A soluble protein fraction containing cytoplasmic esterases was obtained by centrifugation at 1000 g for 5 min. To this soluble protein fraction, DAF-DA was added up to 10 µm and incubated for 90 min at 25°C. After this time, NO was bubbled for 10 s through the reaction medium at a concentration change rate of 0.1 µm s−1 (Ferrer & Ros Barceló, 1999), and fluorescence was determined as described above. The effect of L-NAME (1.0 mm), D-NAME (1.0 mm), aminoguanidine (1.0 mm), PTIO (100 µm) and sodium azide (100 µm) on the quenching of the TFGF was then determined by measuring the basal fluorescence level in the presence of inhibitors.

Quantitative determination of NO

The NO levels during xylem differentiation were determined for thin-walled trans-differentiating mesophyll cells at 72 h of culture. For this, 0.5 ml culture samples (cells and medium) were mixed with 0.5 ml of Griess reagent, and NO levels were determined according to Hevel & Marletta (1994), using a standard curve generated with known concentrations of NaNO2. The NO levels during xylem differentiation were also determined by the haemoglobin assay (Hevel & Marletta, 1994) using 1.0 ml culture samples and 5 µm oxygenated haemoglobin (HbO2).

Effect of PTIO on tracheary element differentiation

The effect of PTIO on the extent of tracheary element formation by trans-differentiating Z. elegans mesophyll cell cultures was tested by adding 100 µm PTIO after 48 h of culture and counting the number of tracheary elements formed at 120 h. The viability of cultured cells was assayed by the fluorescein diacetate method described by Huang et al. (1986).

Results

Production of NO by the differentiating xylem (vascular bundles)

One of the most attractive models for studying the time-course of xylem differentiation is to follow the pattern of vascular differentiation in stems (Shininger, 1979; Mellerowicz et al., 2001). In these organs, the differentiation of xylem elements from procambial cells always follows a continuous centripetal sequence, unlike phloem elements, which follow a centrifugal one. That is, xylem elements which differ as to the extent of cell wall lignification are arranged horizontally from the procambium to the oldest xylem region (Fig. 1). In Z. elegans stems from plants harvested 3 d after the emergence of the first pair of true leaves, the xylem is horizontally arranged in three consecutive cell layers (Fig. 1a), the first layer containing pro-differentiating (living) thin-walled xylem cells (Fig. 1, 1), while the second and third cell layers contain differentiating (Fig. 1, 2) and differentiated (Fig. 1, 3) xylem cells, as judged by the presence of secondary cell wall thickening viewed using polarized light (Fig. 1b).

Figure 1.

Overview of a young Zinnia elegans vascular bundle. Photomicrograph of a young Z. elegans vascular bundle from stems of plants harvested 3 d after the appearance of the first pair of true leaves, as observed either by bright field after staining with toluidine blue (a) or directly by polarized microscopy (b). Images show the presence of a first cell layer containing pro-differentiating (living) thin-walled xylem cells (1), a second cell layer containing differentiating thick-walled xylem cells (2) and a third cell layer containing differentiated thick-walled xylem cells (3), as judged by the presence of the secondary cell wall thickening when viewed by polarized light (b). P, phloem fibres; X, xylem. Bar, 30 µm.

Xylem elements are capable of sustaining NO production, as is revealed (Fig. 2) using NO-fluorescent indicators based on the fluorescein chromophore, which allows real-time biological imaging of NO (Foissner et al., 2000). Thus, when NO production was estimated by CLSM, using DAF-DA as fluorescent probe, in vascular bundles from Z. elegans stems harvested 3 d after the emergence of the first pair of true leaves, TFGF was mainly located in xylem (X) cells regardless of the cell differentiation status (Fig. 2a). However, there was evidence for a spatial NO gradient inversely related to the degree of xylem differentiation, since differentiating xylem cells (arrowheads) clearly manifest a higher capacity for NO production than differentiated xylem cells (arrows). This spatial gradient for NO production is inversely related to the lignification gradient that shows these thick-walled xylem cells, as one may expect from their differentiation status, which is determined by their radial position (Liu et al., 1994). The spatial gradient in NO production by differentiating xylem cells is clearly visible in advanced stages of vascular development (i.e. stems harvested 6 d after the emergence of the first pair of true leaves), where NO production by lignifying xylem cells (Fig. 3a, arrowheads) was greater than NO production by lignified xylem cells (Fig. 3a, arrows). These results confirm that the capability for NO production and cell wall lignification are two inversely related metabolic processes during xylem differentiation. When the values of NO produced by the lignifying xylem, as determined from the green pixel intensity of the TFGF signal, were compared (Fig. 4) with both the lignin content determined by the acetyl bromide method and the phloroglucinol test, a temporally inverse relation was also found.

Figure 2.

Images of nitric oxide (NO) production by a young Zinnia elegans vascular bundle. Image of a young Z. elegans vascular bundle from stems of plants harvested 3 d after the appearance of the first pair of true leaves, after staining with 4,5-diaminofluorescein diacetate (a) and control in the presence of the NO scavenger, 2-phenyl-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide (100 µm) (b), as viewed by confocal laser scanning microscopy. Images show the presence of triazolofluorescein green fluorescence in the first cell layer containing pro-differentiating (living) thin-walled xylem cells (1, asterisk), the second cell layer containing differentiating thick-walled xylem cells (2, arrowheads) and the third cell layer containing differentiated thick-walled xylem cells (3, arrows). P, phloem fibres; X, xylem. Bar, 40 µm.

Figure 3.

Images of nitric oxide (NO) production by a mature Zinnia elegans vascular bundle. Image of a mature Z. elegans vascular bundle from stems of plants harvested 6 d after the appearance of the first pair of true leaves, after staining with 4,5-diaminofluorescein diacetate (a) and control in the presence of the NO-scavenger, 2-phenyl-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide (100 µm) (b), as viewed by confocal laser scanning microscopy. Images show the presence of triazolofluorescein green fluorescence in the cell layer containing lignifying thick-walled xylem cells (arrowheads) and the cell layer containing lignified thick-walled xylem cells (arrows). P, phloem fibres; X, xylem. Bar, 30 µm.

Figure 4.

Temporal inverse relationship between nitric oxide (NO) production and the lignin content in the Zinnia elegans xylem. Photomicrograph of a young (a) and mature (b) Z. elegans vascular bundle after staining with phloroglucinol in stems harvested 3 d and 6 d after the appearance of the first pair of true leaves, respectively. Bar, 100 µm. (c) Values of NO produced by the lignifying xylem, as determined from the green pixel intensity of the triazolofluorescein green fluorescence (TFGF) signal, and lignin content determined by the acetyl bromide method, in young and mature Z. elegans vascular bundles. Fluorescence images were monitored in 25 sections selected at random from 20–30 plants, with 150 vascular bundles being observed in each assay. Bars show SE; n = 150 for TFGF values and n = 5 for lignin content values.

In all the cases, TFGF was totally sensitive to the NO scavenger PTIO (Figs 2b and 3b). Since PTIO did not quench TFGF (Table 1), these results firmly suggest that TFGF was due to NO, and not to other nitrogen oxides, NOx, such as NO2 and N2O3.

Table 1.  Effect of metabolic inhibitors (L-NAME, D-NAME, aminoguanidine and NaN3) and the nitric oxide (NO)-scavenger PTIO on the quenching of the TFGF1
Reaction mediumTFGF (green pixel intensity)
  • Values are means ± SE. Values followed by the same letter are not significantly different at P = 0.05.

  • 1

    L-NAME, NW-nitro-l-arginine methyl ester; D-NAME, Nw-nitro-d-arginine methyl ester; PTIO, 2-phenyl-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide; TFGF, triazolofluorescein green fluorescence.

DAF + NO244 ± 8a
DAF + NO +1.0 mm L-NAME229 ± 10a
DAF + NO +1.0 mm D-NAME250 ± 0a
DAF + NO +1.0 mm aminoguanidine244 ± 3a
DAF + NO +100 µm NaN3250 ± 3a
DAF + NO +100 µm PTIO243 ± 6a

The situation is more complex in pro-differentiating (living) noncollapsed thin-walled xylem cells (Fig. 2a, 1), where a burst of NO production is clearly visible inside the protoplast (Fig. 2a, asterisk). In these cells, TFGF was seen both in the protoplast and in the cell wall. These results suggest that cambial cells, which are predetermined to irreversibly differentiate in xylem elements (Fig. 1, 1), show a burst of NO production which is sustained while secondary cell wall synthesis and cell autolysis are in progress.

Lignifying phloem (phloem fibres) showed similar features to the xylem for NO production (Fig. 3a). However, no other tissues, including the epidermis, cortical parenchyma and pith, showed NO production so that NO production remains characteristic of vascular (predetermined to die) conducting cells.

Production of NO by differentiating xylem cells was partly sensitive to L-NAME, a competitive inhibitor of plant nitric oxide synthases (NOS, EC 1.14.23.39) (Barroso et al., 1999) (Table 2). However, D-NAME, the enantiomer of L-NAME, which was used as negative control (Barroso et al., 1999), was also capable of inhibiting NO production by differentiating xylem cells to almost the same extent. Aminoguanidine (an irreversible inhibitor of inducible NOS) (Barroso et al., 1999) was unable to inhibit TFGF (Table 2), as was the case with sodium azide (Table 2), an inhibitor of nitrate reductase (Yamasaki & Sakihama, 2000). These results suggest that NO production by differentiating xylem cells is unlikely to be catalysed either by an l-arginine-dependent NOS-like enzyme or by nitrate reductase, and they suggest therefore other possible origins for NO synthesis in xylem cells.

Table 2.  Effect of metabolic inhibitors (L-NAME, D-NAME, aminoguanidine and NaN3) and the nitric oxide (NO)-scavenger PTIO on the mean values of the green pixel intensity of the TFGF signal of the differentiating xylem of Zinnia elegans1
Reaction mediumTFGF (green pixel intensity) (n)
  • Values are means ± SE. Values followed by the same letter are not significantly different at P = 0.05.

  • 1

    L-NAME, NW-nitro-l-arginine methyl ester; D-NAME, Nw-nitro-d-arginine methyl ester; PTIO, 2-phenyl-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide; TFGF, triazolofluorescein green fluorescence.

Control238 ± 6a (108)
+1.0 mm L-NAME162 ± 6b (459)
+1.0 mm D-NAME184 ± 9b (99)
+1.0 mm aminoguanidine231 ± 5a (22)
+100 µm NaN3229 ± 7a (42)
+100 µm PTIO  0 ± 0b (52)

Production of NO by tracheary elements

Studies on xylem development (tracheary element differentiation) in plant cell cultures have provided a strong support in the knowledge of xylem differentiation from cambial derivatives, since cultured mesophyll cells can be induced by external stimuli to proceed through temporally controlled metabolic and developmental programs that conclude in the formation of single-cell-derived dead vascular tracheary elements (Fukuda, 1997; Demura et al., 2002).

Trans-differentiating Z. elegans mesophyll cells may be monitored at different developmental stages: undifferentiated mesophyll cells at 24 h of culture (Fig. 5a); thin-walled trans-differentiating mesophyll cells at 72 h of culture (Fig. 5c); thick-walled trans-differentiating (dying) mesophyll cells at 72 h of culture (Fig. 5f); and thick-walled differentiated (died) tracheary elements at 96 h of culture (Fig. 5h).

Figure 5.

Images of nitric oxide (NO) production by trans-differentiating Zinnia elegans mesophyll cells at different developmental stages. Bright-field (a,c,f,h) and fluorescent (b,d,e,g,i,j) images of undifferentiated mesophyll cells (a,b), thin-walled trans-differentiating mesophyll cells at 72 h of culture (c,d,e), thick-walled trans-differentiating mesophyll cells at 72 h of culture (f,g) and thick-walled differentiated tracheary elements (h,i,j), stained with 4,5-diaminofluorescein diacetate in the absence (b,d,g,i) and in the presence (e,j) of 100 µm 2-phenyl-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide, as viewed by confocal laser scanning microscopy. Bar, 15 µm.

For NO production, trans-differentiating Z. elegans mesophyll cells maintain the same property shown by the differentiating xylem from the Z. elegans vascular bundles. Thus, when NO production was again estimated in tracheary elements derived from Z. elegans mesophyll cell cultures by CLSM using DAF-DA as fluorescent probe, TFGF was mainly observed in pro-differentiating thin-walled (Fig. 5c,d) and differentiating (secondary cell wall forming) (Fig. 5f,g) tracheary elements. Mesophyll cells which have not acquired competence for trans-differentiation showed only background TFGF levels (Fig. 5a,b). By using both the Griess and haemoglobin assays, the NO peak during this stage was established at 1.5 µm (Table 3). As occurs in the Z. elegans vascular bundle (Fig. 2), this burst in NO production in tracheary elements was sustained while secondary cell wall synthesis was in progress (Fig. 5h,i). TFGF was again totally sensitive to the NO scavenger, PTIO (Fig. 5e,j), and was therefore due to NO. These results confirm that NO formation by tracheary elements is directly related to the early processes of xylem differentiation, which take place immediately before the late processes of secondary cell wall formation and cell autolysis.

Table 3.  Levels of nitric oxide (NO) produced by thin-walled trans-differentiating mesophyll cells of Zinnia elegans at 72 h of culture, as assayed by the Griess and haemoglobin reagent
ReagentNO (µm)
  1. Values are means ± SE. Values followed by the same letter are not significantly different at P = 0.05.

Griess1.53 ± 0.16a
Haemoglobin1.20 ± 0.60a

That the NO produced by trans-differentiating Z. elegans mesophyll cells is necessary for tracheary element differentiation was demonstrated by using PTIO (Table 4). The addition of this NO scavenger to trans-differentiating Z. elegans mesophyll cells inhibited tracheary element differentiation by 78% while cell viability increased by 51%, as assayed by the fluorescein diacetate method.

Table 4.  The effect of PTIO1 on cell viability and tracheary element formation of trans-differentiating Zinnia elegans mesophyll cell cultures (4 × 105 cells ml−1) assayed after 120 h of culture
ParameterControl cellsPTIO-treated cells (100 µm)
  • Values are means ± SE. Values followed by the same letter are not significantly different at P = 0.05.

  • 1

    PTIO, 2-phenyl-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide.

Viability (%)53 ± 6a80 ± 10b
Tracheary elements (%)45 ± 5a10 ± 3b

Discussion

Lignins are complex cell wall phenolic heteropolymers which result from the oxidative polymerization of the monolignols, p-coumaryl, coniferyl and sinapyl alcohol. Lignins are mainly localized in the impermeable water transport conduits of the xylem and phloem fibres, vascular tissues that are also capable of sustaining NO production as determined both in vascular bundles and in in vitro xylogenesis models. When NO production was estimated in both vascular bundles (Figs 2 and 3) and tracheary elements (Fig. 5) from Z. elegans by CLSM, using DAF-DA as fluorescent probe, NO was mainly located in xylem cells and phloem fibres, regardless of the cell differentiation status. However, there was evidence for a spatial (Figs 2 and 3) and temporal (Fig. 4) NO gradient inversely related to the degree of xylem differentiation, so that the capability for NO production and cell wall lignification are apparently two inversely related metabolic processes. This inverse relation is intriguing since all the branching enzymes, as well as rate-limiting enzymes (Anterola et al., 2002), of the lignin biosynthetic pathway, cinnamate-4-hydroxylase (C4H, trans-cinnamate, NADH: oxygen oxidoreductase [4-hydroxylating], EC 1.14.13.11), p-coumarate-3-hydroxylase (C3H, p-coumarate, NADH: oxygen oxidoreductase [3-hydroxylating]) and coniferylaldehyde-5-hydroxylase (CAld5H, coniferylaldehyde, NADH: oxygen oxidoreductase (5-hydroxylating)), and the lignin-assembling enzyme, peroxidase (hydrogen donor, H2O2: oxidoreductase, EC 1.11.1.7), are haem proteins and therefore possible targets of NO action (Tsai, 1994). This is especially important since any possible metabolic control of these enzymes by the NO synthesized by the developing xylem would enable it to regulate not only the global p-hydroxycinnamyl alcohol pools in lignifying plant cells and the p-hydroxyphenyl–guiacyl–syringyl (H : G : S) ratio for carbon partitioning, but also their rates of polymerization.

However, it is to be expected that the effects of NO on cell wall lignification will be rather complex since, a priori, NO could modulate the activity of lignin biosynthetic enzymes through a combination of effects, including transcriptional regulation, as it is the case of C4H, cinnamoyl-CoA reductase and cinnamyl alcohol dehydrogenase (Polverari et al., 2003; Parani et al., 2004), altering substrate availability, and the direct reaction with enzyme turnover intermediates, as it is the case of C4H and peroxidase (Ferrer & Ros Barceló, 1999; Enkhardt & Pommer, 2000). At present, there are no molecular data available on the interaction of NO with the heme-containing mono-oxygenases, C3H and CAld5H, of the lignin biosynthetic pathway, and the only effects described concern C4H (Enkhardt & Pommer, 2000) and peroxidase (Ferrer & Ros Barceló, 1999; Ros Barcelóet al., 2002a). In both cases, NO reacts with the heme group of these haem proteins causing a noncompetitive inhibition of the enzymes, the inhibition persisting only as long as NO is present. Since the step catalysed by C4H is a rate-limiting step in the lignin biosynthetic pathway (Anterola et al., 2002), it remains to be determined how and to what extent this inhibition alters the rate of carbon allocation in the pathway, and whether this totally justifies the inverse relation found between NO levels and lignins in the developing xylem (Figs 2–4).

In vitro xylogenesis models (Fig. 5) revealed some special and particular features concerning the meaning of the NO produced during xylem differentiation. First of all, the results obtained with this in vitro culture system (Fig. 5) supported the observations made in planta (Figs 2 and 3), and indicated that the NO produced by the differentiating xylem is not a wounding response. Furthermore, trans-differentiating Z. elegans mesophyll cells offer several advantages for studying the role played by the NO produced during xylem differentiation. Thus, when Z. elegans mesophyll cells are cultured in an inductive medium, the first morphological manifestation of differentiation occurs approximately 72 h after isolation, when nascent tracheary elements synthesize the elaborate secondary cell wall between the primary cell wall and the plasmalemma (Fig. 5f). This constitutes a critical point in the ‘way without return’ since a few hours after the secondary cell wall becomes visible, the large central vacuole collapses and cytoplasmic streaming ceases simultaneously (Groover et al., 1997), marking the beginning of a critical event during programmed cell death, the execution of cell death (Groover et al., 1997). Hydrolytic vacuolar contents produce the effective degradation of the protoplast and the simultaneous degradation of nuclear DNA, which can be viewed by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) (Groover et al., 1997; Groover & Jones, 1999). This point at which trans-differentiating mesophyll cells take the ‘way without return’ a few hours prior to the collapse of the vacuole, is marked by a burst in NO production (Fig. 5d), which is sustained while secondary cell wall synthesis and cell autolysis is in progress (Fig. 5g). This suggests that the NO produced by tracheary elements is synthesized at the precise moment at which trans-differentiating mesophyll cells acquired the competence for cell death. These results also explain why pro-differentiating thin-walled xylem cells in vascular bundles (Fig. 1, 1) also exhibited an NO burst (Fig. 2a, 1) which, as occurs in tracheary elements (Fig. 5), is sustained while secondary cell wall synthesis and cell autolysis are in progress.

The results described here demonstrate that pro-differentiating xylem elements are capable of sustaining NO levels in the order of µm (Table 3). Results were obtained by means of three independent and highly selective methods: (1) the Griess reagent, based on the formation of azo compounds during the highly specific reaction of NO with aromatic amines (Hevel & Marletta, 1994); (2) the haemoglobin method, based on the reactivity of NO with oxyhaemoglobin to produce methaemoglobin (Hevel & Marletta, 1994); and (3) the NO-fluorescent indicator, the diamino-fluorescein chromophore (DAF), which reacts specifically with NO to produce the highly fluorescent triazolofluorescein, and which furthermore allows the real-time biological imaging of NO (Foissner et al., 2000). In the case of DAF, the membrane-permeable form of this dye, its diacetate ester derivative (DAF-DA), is taken up by the cells and hydrolysed by cytoplasmic esterases to again form the membrane-impermeable compound, DAF. This is in itself nonfluorescent but reacts with NO in the presence of oxygen to form the highly fluorescent triazolofluorescein, which is trapped in cells (Foissner et al., 2000). These properties of DAF-DA explain the imaging of the NO burst in the protoplast of differentiating thin-walled xylem cells (Fig. 2a, asterisk, and Fig. 5d,g), but do not explain how secondary cell walls (Figs 2a and 3a, arrowheads, and Fig. 5i) can also seen to be capable of showing TFGF. To cast light on this aspect, it is sufficient to say that plant cell walls contain acetyl esterases (Tretyn et al., 1997; Kuchitsu et al., 2002), and the broad range of substrate specificity shown by these hydrolytic enzymes is well known. Thus, the presence of esterases in xylem cell walls or, in their defect, the diffusion of cytoplasmic esterases from collapsing/dying xylem cells, together the dicationic nature of DAF (which will bind electrostatically to the negative charged cell walls), could explain the imaging of NO in this subcellular compartment. Its origin could be diffusion from the protoplast, although the presence of nonenzymatic NO-synthesizing mechanisms in the apoplast have recently been described (Bethke et al., 2004). Caution should be exercised, however, when attempting to ascertain the subcellular localization/distribution of NO when using this dye, since DAF is a single-wavelength probe, and no adjustments can be made for differential accumulation of the probe within/outside the cell (Foissner et al., 2000).

In any case, the imaging of NO in xylem cell walls lends support to our observations that NO modulates activities of lignin-assembling cell wall-located enzymes, such as peroxidases (Ferrer & Ros Barceló, 1999; Ros Barcelóet al., 2002a), and this result could easily explain the inverse relation between NO levels and lignins found in the Z. elegans xylem (Figs 2–4).

Nitric oxide is a gaseous, partly water-soluble, and reactive free radical molecule, which has a relatively short half-life of a few seconds. Thus, NO rapidly reacts with O2 to yield NO2 (2NO + O2 → 2NO2) in a complex reaction that is second-order in NO (k[NO]2 [O2]) (Stamler et al., 1992). The NO half-life depends critically therefore on both the initial NO concentration and the O2 tension in the aqueous phase. NO2 can further react with NO to yield other nitrogen oxides, such N2O3 (NO + NO2 → N2O3), which rapidly decays in aqueous solutions to lead to nitrite and nitrate. This raises the question of whether the NO produced by differentiating xylem tissues is involved in signalling between neighbouring plant tissues, neighbouring plant cells, or the same cell in which NO is generated. The answer to this question is contained in Fick's second law:

χ2 = 4DNOτ1/2

From this equation, it can be established that the length of the boundary layer of NO diffusion (action) (χ) may be as short as 100 µm, if we assume that the diffusion coefficient of NO (DNO) is ≈ 1 × 10−9 m2 s−1 (like other gases in water) and its half-life in the aqueous phase (τ1/2) ≈ 2.5 s. These calculations suggest that the targets for NO action would be exclusively located in the xylem (or phloem) cells in which NO is generated, supporting the view that the NO synthesized by the developing xylem is involved only in xylem-specific events.

There is evidence to support an NO production in plants that is dependent on NOS-like enzymes (Barroso et al., 1999; Chandok et al., 2003; Guo et al., 2003), nitrate reductase (NR, EC 1.6.6.1) (Desikan et al., 2002; Rockel et al., 2002) and other possible nonenzymatic sources (Neill et al., 2003; Bethke et al., 2004). The NOS-like activities catalyse the formation of NO from l-arginine at the expense of NADPH and O2. Plant NOS-like activities are sensitive to both l-arginine analogous and aminoguanidine, but insensitive to d-arginine analogous (Barroso et al., 1999), and if this is regarded as a general rule, one may expect that NO synthesis by the xylem is unlikely to be catalysed by an NOS-like enzyme, since it is partly sensitive to both L-NAME and D-NAME, and insensitive to aminoguanidine (Table 2). On the other hand, NR is the key enzyme of nitrate assimilation in plants. It generates NO from nitrite (the product of nitrate reduction by NR) using NAD(P)H as electron donor (Neill et al., 2003), and it is insensitive to l-arginine analogues. NR is, however, sensitive to N3 (Yamasaki & Sakihama, 2000), but results obtained using this inhibitor (Table 2) also discard an NR-dependent NO production. Thus, the source(s) of NO production in the differentiating xylem remain uncertain.

Nitric oxide is involved in many physiological processes in plants, where it serves as a synchronizing chemical messenger involved in cytotoxicity and programmed cell death (van Camp et al., 1998; Durner & Klessig, 1999; de Pinto et al., 2002; Neill et al., 2003). NO works with O2·–/H2O2 to trigger programmed cell death through a finely balanced NO/O2·–/H2O2 cooperation (Delledonne et al., 2001). It should not be surprising, therefore, that these species also work together during the programmed cell death which characterizes xylem differentiation. Lignifying plant tissues are capable of sustaining both NO (Fig. 2) and O2·– (Ogawa et al., 1997; Ros Barceló, 1998) production and O2·– therefore it is likely that in the differentiating xylem both NO and will react to produce the peroxynitrite anion (ONOO):

NO + O2·– → ONOO

Peroxynitrite is formed at a near diffusion-controlled rate (k = 6.7 × 109 m−1 s−1), and it has been postulated to play a major role in cytotoxicity (Delledonne et al., 2001; Wendehenne et al., 2001), although its real role in programmed cell death is uncertain (Fukuto & Ignarro, 1997). Regardless of this uncertainty, NO does not affect O2·–/H2O2 production by the lignifying xylem (Ferrer & Ros Barceló, 1999) or H2O2 production by isolated plant mitochondria (one of the organelles involved in H2O2 production during programmed cell death) (Yamasaki et al., 2001). This is not surprising since the synthesis of both O2·–/H2O2 and NO in plant cells is simultaneously coordinated in response to environmental/hormonal stimuli with no negative crosstalk between them (Delledonne et al., 1998).

As in animals, most NO effects in plants appear to be mediated by cyclic guanosine monophosphate (cGMP) (Durner et al., 1998). This is a well-established second messenger (intracellular signalling) molecule in plant cells, whose levels are transiently modified in response to external stimuli (Wendehenne et al., 2001; Neill et al., 2003). Concentrations of cGMP are increased by the activity of GC, which synthesizes it from GTP, and are returned to the resting values by the action of phosphodiesterases (Neill et al., 2003). In animal cells, NO activates cGMP production via direct activation of GC (a haem protein), the activation persisting only so long as NO is present (Neill et al., 2003). cGMP-mediated effects are likely to be mediated by cADP-ribose (cADPR) (Durner et al., 1998), a calcium-mobilizing second messenger, whose synthesis is stimulated by cGMP. Nitric oxide selectively regulates Ca2+-sensitive ion channels in plant cells by promoting Ca2+ release from intracellular stores to raise cytosolic-free Ca2+ (García-Mata et al., 2003; Gould et al., 2003). At this point it is necessary to remember that the execution of cell death in xylem elements involves an influx of Ca2+ into the cell (Groover & Jones, 1999), and is manifested by a rapid collapse of the vacuole leading to the release of hydrolytic enzymes. In this scenario, a cell signalling pathway involving NO → cGMP → cADPR → Ca2+ → cell differentiation/vacuolar collapse → cell death is one of the possible metabolic cascades that may be present in the differentiating xylem if NO, as suggested above, plays a certain role in xylem differentiation and cell death. The results obtained by using the NO scavenger, PTIO (Table 4), which inhibited the burst in NO production and the phenomenon of tracheary element differentiation in the Z. elegans mesophyll cell culture system, thus reducing cell death, strongly suggest that this metabolic cascade is likely to be present.

In such a scenario, it can be expected that all these possible crosstalks between the NO produced by the differentiating xylem and the metabolic pathways which are differentially expressed in this vascular tissue, including those which lead to cell death, should be considered far from fortuitous. Doubtless, once the capability of lignifying tissues for producing NO has been demonstrated and the possible targets identified, a challenge for future research in this field will be to identify the exact targets of NO action and to determine (and dissect) the real role played by NO in xylogenesis and cell wall lignification.

Acknowledgements

This work was supported by grants from the Fundación Séneca (project #PI-70/00615/FS/01) and MCYT (BMC2001-0271 and BOS2002-03550). C. G. and L. V. G. R. hold a fellowship (FPU and FPI) from the MECYD and the MCYT, respectively.

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