Levels of phenolic compounds and enzyme activity

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the other for determining water-soluble phenolic acids. The pellet was used ... p-hydroxybenzoic acid (retention time 16.23 min), chlorogenic acid (retention time ...

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Australasian Plant Pathology, 2007, 36, 32–38

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Levels of phenolic compounds and enzyme activity can be used to screen for resistance of sugarcane to smut (Ustilago scitaminea) R. de ArmasA , R. SantiagoB , M.-E. LegazB and C. VicenteB,C A

Department of Plant Biology, Faculty of Biology, Havana University, Havana, Cuba. Department of Plant Physiology, Faculty of Biology, Complutense University, Madrid 28040, Spain. C Corresponding author. Email: [email protected] B

Abstract. Smut is a major disease of sugarcane plants and is caused by the fungus Ustilago scitaminea. Resistance to smut seems to be a multifactorial process. The current criteria for the selection of resistant varieties are applied by discarding sensitive specimens rather than improving resistance mechanisms through analysis of the molecular bases for resistance. This paper correlates the sensitivity or resistance to smut with changes in sugarcane leaf levels of free phenolic compounds, and phenylalanine ammonia-lyase and peroxidase activities, in response to elicitors from U. scitaminea mycelium. The interaction was studied in sugarcane leaf discs obtained from cultivars Barbados 42231, which is susceptible to smut, and Mayar´ı 55–14, which is highly resistant to smut. The elicitors enhanced the levels of hydroxycinnamic and hydroxybenzoic acids mainly in the resistant cultivar. p-Coumaric (from hydroxycinnamic acids) and syringic acid (from hydroxybenzoic acids) were the phenolic acids that showed a major accumulation pattern. Cultivar Mayar´ı 55–14 had high phenylalanine ammonia-lyase activity without accumulation of free hydroxycinnamic acids. The increase in peroxidase activity was important in the defence mechanism but was not a determinant for the defence. Cultivar Barbados 42231 showed low phenylalanine ammonia-lyase activity as accumulation of caffeic acid produced feedback inhibition of the enzyme. A simple model for studying the level of resistance of new sugarcane clones for helping breeding programs is proposed.

Introduction Sugarcane (Saccharum spp. hybrids) is a major commercial crop grown in many developing countries, mainly for the high quantity of sucrose that accumulates in the stalks. Smut is an important disease of sugarcane caused by Ustilago scitaminea. The disease affects plant growth and the juice quality (Mart´ınez et al. 2000). Spore germination occurs on the internode surface and it is followed by the formation of appressoria, mainly on the inner scale of young buds and on the bases of emerging leaves (Waller 1970). Entry into the meristem in the bud occurs between 6 and 36 h after the teliospores are deposited on the surface (Alexander and Ramakrishnan 1980). Hyphal growth occurs throughout the infected plant, but mostly in the parenchyma cells of the lower internodes. In the upper internodes, hyphal growth concludes with the formation of the whip (sori with teliospores). Hyphae do not penetrate into the cells of the scale leaves (Singh and Budhraja 1964); therefore, buds tightly enclosed within scale leaves can escape infection. It has been proposed that varied resistance of sugarcane is determined by several morphological features of buds (Waller 1970). However, other authors have suggested that resistance is based on chemical properties rather than on bud morphology. Resistance to disease seems to be a multifactorial process. The response phase includes accumulation of different compounds such as phytoalexins (i.e. low molecular mass antimicrobial compounds that accumulate at sites of infection); systemic enzymes that degrade pathogens (e.g. chitinases, © Australasian Plant Pathology Society 2007

β-1,3-glucanases and proteases); systemic enzymes that generate antimicrobial compounds and protective biopolymers (e.g. peroxidases and phenoloxidases); biopolymers that restrict the spread of pathogens (e.g. hydroxyproline-rich glycoproteins, lignin and callose); and regulators of the induction or activity of defensive compounds (e.g. elicitors of plant and microbial origin, immune signal from primed plants and compounds, which release immune signals) (Kuc 1990). Resistance to smut has also been associated with the accumulation of free or conjugated polyamines in sugarcane tissues (Legaz et al. 1998; Pi˜no´ n et al. 1999) and with the production of several glycoproteins of juices (Mart´ınez et al. 2000). These glycoproteins affect the cytoplasmic polarity during spore germination (Fontaniella et al. 2002) and impede cell polarisation by inhibiting the protrusion of the germ tube and spore germination, indicating that the inhibition of teliospore germination constitutes a defence mechanism involved in the resistance of sugarcane to smut (Millanes et al. 2005; Legaz et al. 2005). Infection by fungal pathogens produces and changes the role and reaction abilities of phenolic compounds (Sedl´aˇrov´a and Lebeda 2001). Early release of preformed phenolics and their later intensive production after stimulation of phenylpropanoid metabolism are a part of resistance reactions to disease in many plants (Peltonen 1998). Within the central vacuole, pre-existing phenylpropanoids are stored serving as a pool of substances to be incorporated into the cell wall when released to the cytoplasm during the initial stages of plant defence. These 10.1071/AP06077

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processes are dependent on peroxidases and other enzymes in the apoplast that provide phenolic acid esterification. Only during later pathogenesis is de novo synthesis of phenolic compounds switched on, following the transcriptional activation of genes for phenylpropanoid biosynthesis, which is closely associated with phenylalanine ammonia-lyase (PAL) (Guidi et al. 2005). Lignins are formed and used to increase rigidity of cell walls, and phenolics are part of the hypersensitive response (Lebeda et al. 2001). According to Benda (1987), it is not difficult to find resistant varieties of sugarcane to smut. The problem is that it is difficult to find an association between high resistance and desirable agronomic characteristics. The current criteria for the selection of resistant varieties are applied by discarding sensitive specimens rather than improving resistance mechanisms through analysis of the molecular bases for resistance. Any additional criteria being able to promote the distinction between susceptible and resistant varieties would help breeding programs to characterise promising clones. The aim of this work was to relate sensitivity or resistance to smut with changes in concentrations of phenolic compounds and the activity of some associated enzymes, produced by smut elicitors in sugarcane leaves. An additional aim was to propose a simple model to study the level of resistance of new sugarcane clones in order to help breeding programs. Material and methods Plant material Field-grown 12-month-old Saccharum officinarum cv. Mayar´ı 55–14 (resistant to smut) and cv. Barbados 42231 (highly susceptible to smut), were used throughout this work. Discs of 1.0-cm diameter were obtained from central part of the first completely developed young leaves of different stalks. Leaves were submerged in water during disc preparation. Twenty discs were used for each treatment. Elicitor preparation Teliospores of U. scitaminea (20 mg) were isolated from whips collected from diseased Barbados 42231 plants in experimental crops of the National Institute for Sugarcane Investigation (INCA) in Matanzas, Cuba. The collected teliospores were incubated in 200 mL of sterile Lilly and Barnett medium (Lilly and Barnett 1951) at 38◦ C for 5 days. The mycelium formed was harvested, washed with distilled water, lightly dried with filter paper, weighed and ground to a fine powder in liquid nitrogen (3.6 g wet weight). The powder was extracted with 25 mL of 10 mM Tris-HCl, pH 8.8. Following centrifugation (5000g for 10 min at 4◦ C), 20 mL of 80% (v/v) methanol was added to the pellet and the mixture was shaken for 4 h at 38◦ C. After centrifugation, the pellet was washed once with 5 mL methanol and dried under air flow for 2 h. The dried pellet was washed with 10 mM phosphate buffer, pH 6.8, and the pellet was resuspended in 25 mL of the extraction buffer; the mixture was autoclaved (120◦ C for 30 min) and centrifuged. The clear supernatant was used to elicit the response from sugarcane leaves. Effect of smut elicitor on sugarcane leaves Twenty discs of sugarcane leaves (0.5 g fresh weight approximately) were floated on 10 mM phosphate buffer, pH 6.8

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to a final volume of 15 mL, containing 4% isopropylic alcohol for 2 h at 37◦ C using Petri plates in the dark. Then, 0.5 mL of the elicitor solution was added to discs and incubated in the dark from 0 to 72 h or in photoperiodic cycles (50% of light in each cycle) using a growth chamber. When indicated, different quantities of elicitor (from 0 to 1.0 mL) were used and the incubation was performed in darkness for 6 h. Control experiments were performed in the absence of elicitor. Three biological replications were made by using leaf samples from different stalks. Preparation of cell-free extracts from sugarcane leaf discs After incubation with or without elicitor, leaf discs were washed with distilled water and immediately ground to a fine powder in liquid nitrogen. Powder was resuspended in 5 mL of 10 mM phosphate buffer, pH 6.8. Following centrifugation (10 000g for 15 min at 2◦ C), the supernatant was separated into two aliquots: one for assaying enzyme activity and the other for determining water-soluble phenolic acids. The pellet was used for the extraction of phenolic acids soluble in methanol. Extraction and analysis of phenolic acids Water-soluble phenolic acids were extracted from the supernatant partitioned twice in organic phases with 5 mL diethylether : ethylacetate (65 : 35 v/v). Organic phases were mixed and evaporated to dryness in moving air. Residues were redissolved in 0.1 mL acetonitrile and used for high performance liquid chromatography (HPLC) analysis. For the estimation of methanol-soluble phenolic acids, the pellet was extracted with 5 mL 80% (v/v) methanol at 70◦ C for 1 h with continuously shaking, and centrifuged (15 000g for 15 min at 2◦ C). The supernatant was dried in moving air. Dry residues were redissolved in 0.2 mL acetonitrile and used for HPLC analysis. Where indicated, ethylic and methanolic phases were mixed and evaporated together to dryness under air flow. High performance liquid chromatography separation was carried out using a liquid chromatograph (Spectra Physics 8810). Analytical conditions were as follows: column, Tracer Excel 120 ODSB (25 cm × 4.6 mm internal diameter); injection, 10 µL; mobile phase, solvent A: acetonitrile (100%) and solvent B: acetic acid/water (2 : 98, v/v); gradient, from 100% B (7 min), to 25% A + 75% B (35 min and then maintained for another 5 min), to 100% B (50 min); flow rate 0.9 mL/min; temperature, 25◦ C; absorbance unit full scale, 0.005; detector, UV-Vis SP8490 (λ = 270 nm); internal standard, salicylic acid, 0.5 mg/mL. All chemicals for the standards, gallic acid (retention time 6.60 min), protocatechuic acid (retention time 10.46 min), p-hydroxybenzoic acid (retention time 16.23 min), chlorogenic acid (retention time 23.11 min), caffeic acid (retention time 23.73 min), syringic acid (retention time 24.77 min), p-coumaric acid (retention time 27.40 min), ferulic acid (retention time 28.49 min), benzoic acid (retention time 30.01 min) and cinnamic acid (retention time 39.48 min), were purchased from Sigma Chemical Co. (St Louis, MO). Quantitative estimation of each phenolic compound was done by using the slope of the straight line obtained by linear regression from different injected mass of the phenolic compounds and their corresponding area counts.

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expressed as µg of cinnamic acid produced per mg protein per min. Protein was estimated according to Lowry et al. (1951).

Phenolic concentration (µg/g fresh weight)

Assays for determining enzymatic activities Peroxidase (POX) activity was assayed using reaction mixtures containing 2.7 mL 10 mM phosphate buffer, pH 6.8; 10 µL guaiacol; 200 µL hydrogen peroxide and 50 µL of supernatant from cell free extracts. A unit of specific activity was defined as 1.0 unit of absorbance at 470 nm/mg protein.min (Millanes et al. 2005). Control experiments were performed in the absence of hydrogen peroxide. Phenylalanine ammonia-lyase activity was measured using the procedure described by McGhie et al. (1997), with modifications (A. M. Millanes, R. de Armas, R. Santiago, M. E. Legaz and C. Vicente, unpublished data). Reaction mixtures contained 0.6 µL 0.1 M Tris-HCl buffer, pH 8.8, 0.4 mL supernatant from cell free extracts and 2 mL 10 mM L- phenylalanine (Sigma Chemical Co.) as a substrate, in a final volume of 3.0 mL. The reaction was started by adding the substrate and carried out at 37◦ C from 0 to 6 h, measuring the absorbance of the mixture at 30 min intervals, using a Unicam Helios β spectrophotometer (Unicam, Cambridge, UK). The increment of the absorbance at 275 nm in the linear section of each timecourse was used to calculate the amount of cinnamic acid produced and the slope of the straight line after fitting experimental data by linear regression was used as the reaction rate value. Phenylalanine ammonia-lyase activity was then

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Effect of elicitor concentration on accumulation of phenolic acids Different elicitor concentrations produced different responses in both sugarcane cultivars when leaf discs were incubated for 6 h in the dark. For cv. Barbados 42231 (susceptible to smut), the total concentrations of hydroxycinnamic acids (p-coumaric + caffeic + chlorogenic + ferulic acids) and hydroxybenzoic acids (p-hydroxybenzoic + protocatechuic + syringic acids) were only slightly higher than those obtained in the absence of elicitor (control). The concentrations of both groups of phenolic acids analysed in cv. Mayar´ı 55–14 (resistant to smut) were higher than those obtained in cv. Barbados 42231, with the highest concentration of hydroxycinnamic acids at the 0.1 and 0.25 mL elicitor treatments and a continuous increase in the concentration of hydroxybenzoic acids with increasing elicitor concentration (Fig. 1a). PAL activity in this cultivar increases with increasing elicitor concentration, whereas no variation occurred in cv. Barbados 42231 (Fig. 1b). POX activity appears to be regulated differently to PAL activity, as the maximal values

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Volume of elicitor extract (mL) Fig. 1. Concentration of total hydroxycinnamic acids (squares) and total hydroxybenzoic acids (triangles), PAL activity and POX activity at different elicitor concentrations in cv. Barbados 42231 (susceptible) (a, c and e) and cv. Mayar´ı 55–14 (resistant) (b, d and f). Values are the mean of three replicates. Vertical bars represent the standard error of the means.

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of activity of these enzymes were obtained for very different concentrations of the elicitor. Increased POX and PAL activity were observed in response to elicitor in cv. Barbados 42231. In Mayar´ı 55–14, POX activity only increased in the 0.1 mL elicitor treatment (Fig. 1c).

of phenolic acids, principally those from the hydroxycinnamic group. Again, both cultivars showed differences. The smutsusceptible cv. Barbados 42231 showed hydroxycinnamic acids titres similar to those found for the control throughout the timecourse, but the smut resistant cv. Mayar´ı 55–14 always showed higher values than that obtained for the control, when sugarcane discs were incubated in the presence of the elicitors. In both cultivars, titres of hydroxybenzoic acids were very similar in the controls and treatments (Fig. 2a). In parallel with phenolic acid accumulation, the activity of PAL was assayed in both cvv. Mayar´ı 55–14 and Barbados 42231 (Fig. 2b). Maximum PAL activity was detected after 15 h incubation in both cultivars, although higher values were obtained in cv. Mayar´ı 55–14 than in cv. Barbados 42231. Whereas treatment with the elicitors produced higher values of PAL activity than that of controls in cv. Mayar´ı 55–14, in cv. Barbados 42231 the opposite occurred. POX activity was also assayed in both cultivars (Fig. 2c). Higher values of POX activity were detected in cv. Barbados 42231 than in cv. Mayar´ı 55–14. Only slight differences in activity appeared between controls and elicitor treatments except in the 24-h incubation of sugarcane discs of cv. Barbados 42231, where POX activity appeared to be inhibited by the elicitor.

Timecourse of accumulation of phenolic acids

Phenolic concentration (µg/g fresh weight)

The timecourse study was conducted in continuous darkness or in light and dark cycles. Similar patterns of phenolic acid accumulation were detected in both treatments and for this reason, results obtained in light and dark cycles are not shown. The phenolic acid accumulation was studied in the aqueous phase of cell-free extracts prepared in phosphate buffer and extracted with organic solvents and in the aqueous-insoluble residue extracted with methanol. The concentration of watersoluble phenolic acids represented ∼6% of total phenolic acids found, where chlorogenic, caffeic and p-coumaric acid (data not shown) were the most representative compounds. For this reason, both phenolic acid fractions extracted were analysed together. Fig. 2a shows the timecourse of phenolic acid accumulation produced in the dark both in cvv. Mayar´ı 55–14 and Barbados 42231. High levels of phenolic acids were obtained after 15 h of incubation; longer time values produced lower concentrations 70

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Time (h) Fig. 2. Timecourse of accumulation of total hydroxycinnamic acids (squares) and total hydroxybenzoic acids (triangles), PAL activity and POX activity in cv. Barbados 42231 (susceptible) (a, c and e) and cv. Mayar´ı 55–14 (resistant) (b, d and f). Unfilled symbols and bars represent the controls and filled symbols and bars represent samples treated with elicitor. All values are the mean of three replicates. Vertical bars represent the standard error of the means.

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Table 1 shows the concentration range of the major phenolics detected in sugarcane discs incubated over 72 h with or without elicitors in phosphate buffer. From the hydroxycinnamic acids series, the concentration of p-coumaric acid was the highest. From the hydroxybenzoic acids series, the concentration of p-hydroxybenzoic was the lowest. The timecourse of p-coumaric and syringic acid accumulation in cvv. Barbados 42231 and Mayar´ı 55–14 are shown in Fig. 3. The resistant cultivar displayed higher concentrations of both phenolics with lower values when incubations were performed in the absence of an elicitor. In the susceptible cv. Barbados 42231, similar concentration values were observed in the presence as well as in the absence of elicitors. Fig. 4 shows the concentration of both caffeic and chlorogenic acids obtained after a 15-h incubation in the presence of elicitors. Whereas the concentrations of both Table 1.

phenolics were low in sugarcane discs incubated with elicitor in the susceptible cv. Barbados 42231, the concentrations were high in the resistant cv. Mayar´ı 55–14. Discussion It is well known that elicitor molecules derived from fungi are able to evoke extensive biochemical changes and transcriptional activation of many defence-related genes in plants (Darvill and Albersheim 1984). Soluble elicitors produced by smut caused changes in the titre of phenolic acid compounds and the activity of PAL and POX when they interacted with sugarcane leaf discs. The principal change was accumulation of hydroxycinnamic acids in cv. Mayar´ı 55–14 (highly resistant to smut); however, in both cultivars, low concentrations of elicitors (0.1 mL containing 0.03 mg protein) enhanced the level of hydroxycinnamic acids after incubation for 6 h (Fig. 1a). This is a typical plant defence reaction (El Modafar et al. 2000; Sedl´aˇrov´a and Lebeda 2001). High elicitor concentrations diminished free phenolic acid levels in both cultivars. The main difference between the cultivars was that PAL activity only increased in the smut-resistant cultivar in response to the elicitor. When this activity increased, other defence compounds including lignin and phytoalexins such as isoflavonoids and coumarins would be synthesised conferring resistance to smut (Dixon and Paiva 1995). Phytoalexins are antimicrobial agents that are produced in plants in response to the infection and depend on the recognition of a pathogen elicitor (Morrissey and Osbourn 1999). Although a transient increase in hydroxycinnamic acid was observed at the 15-h timepoint, no significant accumulation of hydroxycinnamic acids was detected overall, possibly because PAL catalyses the conversion of L-phenylalanine to trans-cinnamic acid, in the first step of the phenylpropanoid pathway. Another possibility to explain the decrease in free phenolic acid concentration is the conjugation of

Concentration range of individual phenolics in sugarcane leaves in timecourse experiments See also Fig. 2a

Phenolic acids

Concentration range (µg/g leaf fresh weight)

Hydroxycinnamic acids p-Coumaric acid Caffeic acid Chlorogenic acid Ferulic acid

10.6–28.5 1.4–6.5 1.5–16.3 1.9–11.8

Hydroxybenzoic acids p-Hydroxybenzoic acid Protocatechuic acid Syringic acid

Syringic concentration (µg/g fresh weight)

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0.2–1.9 0.2–7.2 1.2–4.6

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Time (h) Fig. 3. Timecourse of accumulation of p-coumaric acid (squares) and syringic acid (triangles) in cv. Barbados 42231 (susceptible) (a and c) and cv. Mayar´ı 55–14 (resistant) (b and d). Unfilled symbols represent the controls and filled symbols represent samples treated with elicitor. All values are the mean of three replicates. Vertical bars represent the standard error of the means.

Phenolic concentration (µg/g fresh weight)

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Fig. 4. Concentration of caffeic acid and chlorogenic acid after 15 h of incubation with 0.5 mL smut-elicitor in cv. Barbados 42231 (susceptible) (a) and cv. Mayar´ı 55–14 (resistant) (b). Unfilled bars represent the controls and filled bars represent samples treated with elicitor. All values are the mean of three replicates. Vertical bars represent the standard error of the means.

free hydroxycinnamic and hydroxybenzoic acids to polyamines. Such conjugates have been related to the sensitivity of some sugarcane cultivars to smut (Legaz et al. 1998). Another studied defence reaction was related to changes in POX activity. Increases in POX activity were detected in cv. Barbados 42231 at all elicitor concentrations tested, whereas an increase in POX activity was only achieved in cv. Mayar´ı 55–14 at the lowest elicitor concentration (Fig. 1c). POX activation is related to lignin accumulation and conversion of several phenolics into oxidised or polymerised compounds (Lebeda et al. 2001). When the interaction time between leaves and elicitors increased, differences between both the susceptible and resistant cultivars were greater (Fig. 2). PAL activity (Fig. 2b) and the concentration of free hydroxycinnamic acids (Fig. 2a) were significantly, but transiently, enhanced in cv. Mayar´ı 55–14 in the 15-h treatment; however, the levels decreased again at later timepoints. It had been demonstrated that high resistance to smut in sugarcane was associated with the possibility of maintaining high levels of PAL activity. The increase in the concentration of free phenolic compounds, PAL activity and POX activity in a short time period (at least to 20 h) was detected in both cultivars, but only the resistant cv. Mayari 55–14 was able to maintain high levels of PAL activity for longer periods of time (Fig. 2b). This fact could be related to the high levels of caffeic acid and its derivate, chlorogenic acid, that are maintained in cv. Barbados 42231 but not in cv. Mayar´ı 55–14 (Fig. 4). Accumulation of caffeic acid produces feedback inhibition of PAL activity according to A. M. Millanes, R. de Armas, R. Santiago, M. E. Legaz and C. Vicente (unpublished data). The fact that incubation times longer than 20 h enhanced PAL activity in cv. Mayar´ı 55–14 (Fig. 2b) without an apparent increase in accumulation of hydroxycinnamic acids, could be due to a very fast metabolic conversion of newly produced hydroxycinnamic acids to more structurally complicated phytoalexins (Franc¸a et al. 2001). Phytoalexins are not present in healthy plants but are synthesised in response to pathogen attack (Morrissey and Osbourn 1999). The conjugation of free phenolic acids to other compounds is another possibility that may explain the lack of apparent increase in hydroxycinnamic acids. Conjugation of phenolic compounds has been shown in sugarcane by de Armas et al. (1999) and in relation to sugarcane smut resistance by Legaz et al. (1998).

p-Coumaric acid was the major hydroxycinnamic acid detected (Table 1) and its timecourse accumulation (Fig. 3) conditioned the timecourse accumulation of total free phenolic acids (Fig. 2a). This phenolic acid is an important precursor for the synthesis of other hydroxycinnamic and hydroxybenzoic acids and participates in reinforcing the cell wall with the formation of ester–ether bridges (Lam et al. 1992). It may be concluded that monitoring changes in leaf phenolic compound concentrations and PAL and POX activities in response to soluble elicitors extracted from U. scitaminea mycelium can afford reliable analyses of the resistance of sugarcane to smut. A resistant cultivar needs to maintain a high level of PAL activity without accumulation of free hydroxycinnamic acids. Increase in POX activity is important in the defence mechanism but it is not a determinant for the defence mechanism. It is possible to use this model for the screening of smut resistance levels of different sugarcane cultivars and it would help breeding programs to characterise promising clones. Acknowledgements This work has been supported by grants from the Secretar´ıa de Estado de Universidades e Investigaci´on SAB2003–0183 and the Ministerio de Educaci´on y Ciencia (Spain) BFI2003–06234. We gratefully acknowledge the excellent technical assistance of Mrs Raquel Alonso.

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Received 26 April 2006, accepted 23 September 2006

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