Journal of Experimental Botany, Vol. 58, No. 11, pp. 2733–2744, 2007 doi:10.1093/jxb/erm138 Advance Access publication 3 July, 2007 This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER
Xylella fastidiosa disturbs nitrogen metabolism and causes a stress response in sweet orange Citrus sinensis cv. Peˆra Ru´bia P. Purcino1,2, Camilo La´zaro Medina3, Daniel Martins de Souza4, Fla´via Vischi Winck4, Eduardo Caruso Machado5, Jose´ Camilo Novello4, Marcos Antonio Machado6 and Paulo Mazzafera1,* 1
Departamento de Fisiologia Vegetal, Instituto de Biologia, Universidade Estadual de Campinas, CP 6109, 13083-970, Campinas, SP, Brazil 2 Universidade Federal Rural do Rio de Janeiro, Km 7 Rodovia BR 465, 23850-000, Serope´dica, RJ, Brazil 3
Conplant, Rua Francisco Andreo Aledo, 22, 13084-200, Campinas, SP, Brazil Departamento de Bioquı´mica, Instituto de Biologia, Universidade Estadual de Campinas, CP 6109, 13083-970, Campinas, SP, Brazil 5 Instituto Agronoˆmico de Campinas, Centro de Ecofisiologia e Biofı´sica, Av. Bara˜o de Itapura 1481, CP 28, 13012-970, Campinas, SP, Brazil 6 Instituto Agronoˆmico de Campinas, Centro APTA Citros Sylvio Moreira, CP 04, 13490-970, Cordeiro´polis, SP, Brazil 4
Received 21 February 2007; Revised 18 May 2007; Accepted 25 May 2007
Abstract Xylella fastidiosa (Xf) is a fastidious bacterium that grows exclusively in the xylem of several important crop species, including grape and sweet orange (Citrus sinensis L. Osb.) causing Pierce disease and citrus variegated chlorosis (CVC), respectively. The aim of this work was to study the nitrogen metabolism of a highly susceptible variety of sweet orange cv. ‘Peˆra’ (C. sinensis L. Osbeck) infected with Xf. Plants were artificially infected and maintained in the greenhouse until they have developed clear disease symptoms. The content of nitrogen compounds and enzymes of the nitrogen metabolism and proteases in the xylem sap and leaves of diseased (DP) and uninfected healthy (HP) plants was studied. The activity of nitrate reductase in leaves did not change in DP, however, the activity of glutamine synthetase was significantly higher in these leaves. Although amino acid concentration was slightly higher in the xylem sap of DP, the level dropped drastically in the leaves. The protein contents were lower in the sap and in leaves of DP. DP and HP showed the same amino acid profiles, but different proportions were observed among them, mainly for asparagine, glutamine, and arginine. The polyamine putrescine was found in high concentrations
only in DP. Protease activity was higher in leaves of DP while, in the xylem sap, activity was detected only in DP. Bidimensional electrophoresis showed a marked change in the protein pattern in DP. Five differentially expressed proteins were identified (2 from HP and 3 from DP), but none showed similarity with the genomic (translated) and proteomic database of Xf, but do show similarity with the proteins thaumatin, mucin, peroxidase, ABC-transporter, and strictosidine synthase. These results showed that significant changes take place in the nitrogen metabolism of DP, probably as a response to the alterations in the absorption, assimilation and distribution of N in the plant. Key words: Amino acids, citrus variegated chlorosis, nitrate reductase, polyamines, proteases, xylem sap, Xylella fastidiosa.
Introduction Xylella fastidiosa (Xf) is a fastidious bacterium causing several diseases in economical important crops species, such as grape (Vitis vinifera) and citrus (Citrus sinensis) (Hopkins, 1989; Purcell and Hopkins, 1996).
* To whom correspondence should be addressed. E-mail:
[email protected] ª 2007 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
2734 Purcino et al.
Xylella fastidiosa has caused important economic losses in the Brazilian citrus industry where it is known as citrus variegated chlorosis (CVC), first detected in Brazil in the mid-1980s (Rossetti et al., 1990) and since then has spread to all citrus-growing areas in Brazil and South America (Carvalho et al., 1995). The economical importance of CVC has led to complete genome sequencing of Xf (Simpson et al., 2000). It has been suggested that the primary mechanism of pathogenicity of Xf is occlusion of the xylem vessels by the biofilm formed by the bacteria (Souza et al., 2006). This mechanism seems to explain satisfactorily most of the symptoms shown by infected plants, however, biosynthesis of toxins and alterations in hormonal levels may also be involved in the pathogenicity of Xf (Hayward and Mariano, 1997). Several genes coding for proteases were found in the Xf genome (Simpson et al., 2000) as well as one Zn-protease and two serino-proteases that were identified in a proteomic analysis of proteins excreted by Xf cultivated in vitro (Smolka et al., 2003). Proteases might facilitate the lateral movement of the bacteria in the xylem vessels, by digesting protein of the cell wall and increasing the pit diameter (Simpson et al., 2000). In this process, enzymes capable of degrading the cell wall carbohydrates might also be involved in pit enlargement (Simpson et al., 2000). However, proteases might have a nutritional importance for Xf growth in the xylem by releasing small peptides as a source of N (Smolka et al., 2003). Xylella fastidiosa grows exclusively in the xylem and although the xylem sap contains a diversity of compounds such as amino acids, organic acids, and inorganic nutrients, they are usually found in low concentrations, limiting bacterial growth (Purcell and Hopkins, 1996). Glutamine (Gln) and asparagine (Asn) are the main amino acids in the xylem sap of plants (Lea et al., 2007) and for this reason they have been included in several artificial media for Xf growth (Davis et al., 1980; Chang and Donaldson, 1993; Lemos et al., 2003; Almeida et al., 2004; Leite et al., 2004). In addition to these amino acids, Xf appears to have a demand for glucose or organic acids, although the relative importance of each source of C is still under dispute. Very little is known about the nitrogen (N) composition of the Citrus xylem sap. Asn and Gln appear to be the main amino acids in the sap transported from the roots to the shoot of Satsuma mandarins (C. unshiu) (Tadeo et al., 1984). However, the amino acid composition and content seem to vary in the xylem sap of citrus according to the period of the year (Culian˜ez et al., 1981; Ramamurthy and Lu¨dders, 1982; Tadeo et al., 1984). On the other hand, stresses caused by diseases (young-tree decline) seem to enhance the content of amino acids in citrus leaves, particularly arginine (Arg) and praline (Pro). However, no relationship could be established between amino acid
change and the presence of Xf in leaves of Prunus persica (Gould et al., 1991). Pro and Arg increased in the leaves of sweet orange cv. Peˆra grafted on Rangpur lime (C. limonia) in the initial stages of CVC development (Medina, 2002). The increase of Arg content in leaves of sweet orange under stress has been suggested as a detoxification process, where the decrease of glucose content due to photosynthesis limitation cause an accumulation of NHþ 4 to toxic levels (Rabe and Lovatt, 1986). Such a relationship between Arg biosynthesis and NHþ 4 accumulation has been reported by other authors (Lovatt, 1986; Sagee and Lovatt, 1991). However, low levels of NHþ 4 were detected in leaves of sweet orange Peˆra and no variation was observed in water-stressed or non-stressed plants whether infected or not with Xf (Medina, 2002). To our knowledge, there is no information in the literature on the enzymes of N metabolism in sweet orange infected by Xf. The NO 3 transported in the xylem vessels to the leaves is sequentially reduced to nitrite and NHþ 4 by the enzymes nitrate reductase (NR) and nitrite reductase, respectively (Lea et al., 1990). Then NHþ 4 - N will be incorporated by glutamine synthetase (GS, EC 6.3.1.2) into glutamate to form Gln using NHþ 4 and glutamate as substrates (Lea et al., 1990). Much of the Gln is then used for the biosynthesis of other amino acids, mainly Asn, which can also be transported in the xylem sap (Lea et al., 1990). The aim of this investigation was to study N metabolism in sweet orange ‘Peˆra infected with Xf, by analysing nitrogen compounds and activities of proteases, NR, and GS in the xylem sap and leaves of healthy (HP) and diseased plants (DP). In addition, proteins extracts from the petiole of HP and DP were separated by 2D-PAGE electrophoresis aiming to reveal differentially expressed proteins in HP and DP and their relationship with N metabolism and stress response.
Materials and methods Plant material The variety sweet orange ‘Peˆra’, which is highly susceptible to Xf, was grafted onto ‘Rangpur’ lime (C. limonia Osbeck) and infected with Xf by grafting with infected budwoods (Nunes et al., 2004). Healthy and infected plants were kept in a greenhouse in 100 l barrels containing a mixture of soil, manure and sand (3:1:1, by vol.). The greenhouse at the Centro de Citricultura Sylvio Moreira, Instituto Agronoˆmico de Campinas, Cordeiro´polis (2234’ S; 4734’ W; 689 m altitude), SP, Brazil, was completely closed to prevent the passage of insects. When these plants were approximately 5-year-old they were used in the experiments. The plants were watered by a drip irrigation system and fertilized regularly, according to the recommendations for citrus (Grupo Paulista de Adubacxa˜o e Calagem para Citros, 1994). All sap and leaf samples were collected from 2–6 July 2004, from 08.00–10.00 h.
Nitrogen metabolism in citrus infected with Xylella fastidiosa 2735 Xylem sap collection Sap was collected from the apical part (3–5 expanded leaves) of branches from DP and HP. Because of the obstruction of the xylem by the bacteria a pressure pump was used (Gould et al., 1991). The branches were removed with a razor blade and immediately mounted in the pressure chamber. Then, the cut surface was washed with distilled water, blotted dry with absorbent paper and the gas released to increase the pressure in the chamber. Once sap bleeding was observed at the cut stem the pressure was increased up to 10% of the water potential observed at bleeding and then held for sap collection. The sap of several plants was collected until a volume of approximately 1 ml was reached. Five 1 ml replicates were obtained for HP and DP. Collections were carried out between 08.00–10.00 h, when the water tension in the xylem is still not high and transpiration is still reasonable in the DP (Medina, 2002). In the HP the sap was abundant even at low pressures (3–5 bars) while for DP pressures up to 15 bars were necessary. The withdrawn sap was collected with a capillary and maintained on ice during the collection. In the laboratory, the presence of Xf in the saps was tested by PCR. The sap was previously purified with the InstaGene Matrix (Bio-Rad). Two sets of specific primers were used to detect Xf in RT-PCRs (Missanvage et al., 1994; Pooler and Hartung, 1995). RTPCR was carried out in five steps: (step 1) 94 C for 3 min, (step 2) 94 C for 1 min, (step 3) 60 C for 1 min, (step 4) 72 C for 1.5 min (repeated 30 times from steps 2 to 4), (step 5) 72 C for 5 min. The PCR contained 25 ll H2O, 2.5 ll 103 buffer, 2.4 mM Mg2+, 0.25 mM each DNTP, 15 ng of each primer in 0.5 ll H2O, 1 unit Taq polymerase, and 20 ll purified sap. The remaining sap was centrifuged at 4000 g to eliminate the bacteria in the samples, and then stored at –80 C for further analyses. Enzyme activities For GS activity, approximately 1 g of leaves was ground with liquid N2, then extracted with cold (4 C) 5 ml 0.1 M Na-phosphate buffer, pH 7.5, containing 0.5 mM MgCl2.6H2O, 10 mM EDTA, 1 mM DTT, 0.4 mM PMSF, and 100 mg PVPP. The extract was centrifuged 20 min at 16 000 g at 4 C and the supernatant recovered for GS assay (O’Neil and Joy, 1974). NR activity was determined in vivo (Carelli et al., 1990). Leaf discs (1.2 cm in diameter) were infiltrated under vacuum for 2 min with 5 ml of 100 mM Na-phosphate buffer, pH 7.5, containing 25 mM KNO3 and 1% isopropanol. The leaf discs were maintained for 30 min at 35 C in the dark, under agitation, and then boiled for 5 min. The enzyme activity was determined by the amount of NO2 released by the discs in the incubation media after boiling. Samples of 2 ml were mixed 1:1 with 1% sulphanilamide in a mixture of 2.4 M HCl containing 0.02% N-1-naphthyl-ethylene amine. Absorbance was recorded at 540 nm. To measure protease activity azocasein was used as substrate instead of azogelatin (Jones et al., 1998). The leaves were powdered in liquid N2 and then proteases extracted with 100 mM TRIS–HCl, pH 7.5, followed by centrifugation. The reaction was carried out in Eppendorf tubes by initially mixing 50 ll leaf extract or xylem sap with 450 ll 25 mM TRIS–HCl, pH 7.5. After 10 min of preincubation at 40 C, 200 ll 1% azocasein was added to the reaction and incubation proceeded for 6 h at the same temperature. Then, 350 ll was withdrawn from each tube and mixed with 150 ll 10% trichloroacetic acid. After 20 min at room temperature the mixture was centrifuged and the absorbance of the supernatant measured at 440 nm. þ NO 3 , NH4 , amino acids, soluble proteins, and polyamines The same extracts obtained for GS activity were used to measure total soluble proteins (Bradford, 1976), NO 3 (Cataldo et al., 1975),
NHþ 4 (Sodek and Lea, 1993) and soluble amino acids (Cocking and Yemm, 1954). In the sap, the measurements of these substances were made after centrifugation to eliminate the bacteria. Qualitative analyses of amino acids were carried out by HPLC (Shimadzu) with fluorimetric detection after derivatization with o-phthalaldehyde (Jarret et al., 1986). A Spherisorb ODS-2 C18 column was used with 0.8 ml min1 flow rate for a linear gradient formed by solution A, 65% methanol and solution B, pH 7.5 phosphate buffer (50 mM sodium acetate, 50 mM disodium phosphate, 1.5 ml acetic acid, 20 ml tetrahydrofuran, and 20 ml methanol in 1.0 l H2O). The gradient was the proportion of solution A from 20% to 28% between 0 min and 5 min, from 28% to 58% between 5 min and 35 min, from 58% to 75% between 35 min and 40 min, 75% to 95% between 40 min and 56 min, 95% to 96% between 56 min and 60 min, and 96% to 100% between 60 min and 61 min. The amino acids eluting from the column were monitored by a Shimadzu fluorescence detector operating on a 250 nm excitation wavelength and a 480 nm emission wavelength. Twenty microlitres of the amino acid solution and 40 ll of the OPA solution were mixed and, after 2 min, 20 ll of the mixture were injected into the HPLC. To measure proline in the sap the samples were derivatized with the AccQ-Fluor Reagent Kit (Waters) following the manufacturer’s instructions and then analysed by HPLC. In the leaves, Pro was measured with ninhydrin (Ringel et al., 2003). Polyamines were detected by HPLC (Hanfrey et al., 2002). Protein extraction for two-dimensional gel electrophoresis (2D) Proteins of a 1 ml sample of sap were precipitated in 2 ml of cold acetone:ethanol:acetic acid solution (50:49.9:0.1 by vol.) at –20 C, overnight. Precipitated proteins were dissolved in 50 ll of 10 mM TRIS (pH 8.8), 100 mM DTT, 5 mM EDTA, and 1 mM PMSF, boiled for 3 min and stored at –70 C. An aliquot of each of the resulting protein extracts was used to determine the protein concentration using a Bradford protein assay kit (Bio-Rad). The protocols for the 2D electrophoresis are as described in Smolka et al. (2003). Analyses were carried out in triplicate. Protein identification by mass spectrometry Proteins were identified by mass spectrometry using Q-TOF equipment (Applied Biosystems). The protein spots were excised from 2D gels and submitted to in-gel digestion for the extraction of peptides, according to Schevechenko et al. (1996). The mass spectra were obtained by using a hybrid Q-ToF mass spectrometer (Q-ToF Ultima, Micromass, Manchester, UK) with a Zspray source operating in the positive mode. The ionizination conditions include capilar voltage of 2.3 kV, cone and lens voltage RF1 of 30 V and 100 V, respectively, and collision energy of 10 eV. The temperature of the source was –70 C and the nitrogen gas on the cone with the flux of 80 l h1. Argon gas was used to refrigerate the collision and fragmentation of ions in the collision cell. The external calibration was done with sodium iodide for a mass scale from 50 m/z to 3000 m/z. All spectra were acquired with the ToF analyser in V mode (ToF kV¼9.1) and MCP voltage of 2150 V. By using the BLASTX tool (http://www.ncbi.nlm.nih.gov/ BLAST/) the peptide sequences obtained were compared with protein sequences in public databanks (NCBI), using the protein database of the Brazilian Xf proteome project (http://www. proteome.ibi.unicamp.br/index-xylella.htm), the translated nucleotide sequences of the CitEST database of Citrus expressed sequence tags (http://biotecnologia.centrodecitricultura.br/), and the translated sequences of the Brazilian Xf genome project (http://aeg.lbi.ic. unicamp.br/xf/).
2736 Purcino et al. Statistics Data were analysed by ANOVA and means were compared by the Tukey test at 5% significance.
Results Confirmation of Xf infection
2,0
NO31- (mg.g-1 fresh weight)
NO 3 , enzyme activities, proteins, amino acids, and polyamines The analysis of the concentration of NO 3 showed the accumulation of this nutrient in the leaves and in the xylem sap of DP (Fig. 2A, B). However, using colorimetric and HPLC methods only traces of NHþ 4 could be detected in leaves but no positive results in the sap samples. Despite the higher concentration of NO 3 in DP, the activity of NR did not differ between DP and HP plants (Fig. 3A). On the other hand, GS activity was significantly higher in DP (Fig. 3B). Besides a reduction in the protein content in the xylem sap and leaves (Fig. 3C, D), DP also showed a marked increased level of protease activity in the xylem sap and leaves (Fig. 3E, F). Although not statistically significant, there was a clear trend for a lower content of amino acids in the leaves of DP (Fig. 4). On the other hand, the amino acid content was higher in the sap of the same plants (Fig. 4A). Proline (Pro) content in the leaves was similar in DP and HP plants (Fig. 4C). Pro was almost undetectable in the sap. HPLC analysis showed aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), serine (Ser), glutamine (Gln), and arginine (Arg) as the main amino acids in the xylem saps (Fig. 5A). Other amino acids were in very low concentration and are not shown. Significant differences were observed for Asn and Arg. Asn showed the highest decrease from 40.3 mol% in HP to 25.7 mol% in the DP. By contrast, Arg showed an increase in DP, from 12.7 mol% compared with less than 1 mol% in HP. HPLC analysis of amino acids in leaves of DP and HP are shown in Fig. 5B. In the leaves, the main amino acids detected were Asp, Glu, Asn, Ser, Gln, Arg, and Ala, and
Fig. 1. RT-PCR reactions for the detection of Xf in the xylem sap collected from healthy and diseased plants. Markers are shown on bp ¼ base pairs.
A leaves a
1,6
1,2
b
0,8
0,4
0,0 160
NO31- (µg.mL-1 xylem sap)
The yield of sap exudation in the plants infected with Xf was very low because of the obstruction of the xylem vessels by the bacteria. Therefore, samples taken from several plants were mixed until 1 ml was obtained. In order to verify the presence of Xf in these composed samples, PCR assays were carried out using two sets of primers specific for the detection of Xf (Missanvage et al., 1994; Pooler and Hartung, 1995). Both primers were used in the same reactions and therefore two bands of amplification were expected in the sap from DP, as shown in Fig. 1. At the same time, no bands were observed in the sap from HP.
B xylem sap a
120 a 80
40
0 Healthy Plant
Diseased Plant
Fig. 2. Nitrate concentration in the leaves and xylem sap of healthy and diseased plants infected with Xf. Bars indicate standard deviation of five replicates. Different small letters indicate statistical significance at 5% Tukey between diseased and healthy plants.
the others were below 5 mol% and are not shown. The most contrasting differences were found in Gln and Arg (Fig. 5B). While Arg concentration was higher in DP, Gln concentration was lower in these plants. HPLC analysis showed that only putrescine was found in appreciable amounts in the tissues of DP whose concentrations were 15.3 lg g1 and 37.7 lg g1 fresh weight in the petiole and leaves, respectively. Polyamines
A
5
a
4
a
3 2 1 0 300
Healthy Plant
C
250
GS (µmol GHA.g-1 FW.h-1)
6
Diseased Plant
b
200 150 100 50 Healthy Plant
E
b
100 50 0
12
Healthy Plant
D
Diseased Plant
leaves a
10
b
8 6 4 2
80 60 40 20 Healthy Plant
Healthy Plant 140
xylem sap
100
0
a
150
Diseased Plant
Protease (abs x 100)
Protease (abs x 100)
120
B
200
0
0
140
250
14 xylem sap a
mg protein.g-1 leaf FW
µg protein.ml-1 xylm sap
NR (µmol NO2- .g-1 FW.h-1)
Nitrogen metabolism in citrus infected with Xylella fastidiosa 2737
Diseased Plant
120
F
leaves
Diseased Plant
a
100 80
b
60 40 20 0
Healthy Plant
Diseased Plant
Fig. 3. Activities of the enzymes nitrate reductase (A), glutamine synthetase (B), and protein (C, D) contents and protease activity (E, F) in the xylem sap (C, E) and leaves (D, F) of healthy and diseased plants infected with Xf. Bars indicate standard deviation of five replicates. Different small letters indicate statistical significance at 5% Tukey between diseased and healthy plants.
were undetectable in the leaves of HP while traces (1.32 lg g1 fresh weight) were detected in the petioles. Proteome comparison of xylem sap The patterns of protein profile in 2D electrophoresis gels stained with silver nitrate are shown in Fig. 6A and B. Besides differences in the intensity of several spots, it may be observed that DP has more low mass proteins and fewer high molecular mass proteins. Even when doubling the amount of proteins loaded on the gels, many fewer protein spots could be observed with CB staining, but some of the proteins which showed a marked difference in terms of spot intensity remained and six were selected for sequencing (Fig. 6C, D). Five spots were successfully sequenced and identified but they did not have similarities with Xf sequences. These peptides were similar to mucin, strictosidine synthase, ABC-transporter protein, peroxidase, chitinase, and thaumatin (Table 1).
Discussion NO 3 , enzyme activities, proteins, amino acids, and polyamines Figure 7 is a scheme summarizing all the results obtained in this study and is intended to be a guide in the discussion. NO 3 accumulated in the leaves and in the xylem sap of DP, and only a trace of NHþ 4 was detected in the leaves but not in the sap samples. The higher concentration of NO 3 in the DP might be a consequence of an altered N metabolism in the roots, with reduced incorporation in amino acids. The transport of NO 3 from the roots to other parts of the plants depends on the NO 3 assimilation which depends on the NO concentration in the soil, a process 3 highly controlled by a feedback mechanism related to the accumulation of amino acids, mainly Gln (Gojon et al., 1998). Therefore, higher NO 3 accumulation in DP might be a consequence of alterations in this complex
2738 Purcino et al. 60
A xylem sap
A xylem sap
Diseased plant
a
180 a
140
Healthy plant
Amino acids (mol%)
Amino acids (ηmoles.ml-1 xylem sap)
220
100
60
*
40
20
*
20 0
500
Healthy Plant
Diseased Plant
0 Asp
B leaves 60
200
b
100
B leaves
Gln
Arg
*
40
Healthy plant
20
*
0
Proline (µmol.g-1 FW)
Ser
Diseased plant
300
40
Asn
a
Amino acids (mol%)
Amino acids (ηmoles.g-1 leaf FW)
400
Glu
Healthy Plant
C
Diseased Plant
a a
0 Asp
Glu
Asn
Ser
Gln
Arg
Ala
Fig. 5. Amino acid composition of the xylem sap (A) and leaves (B) of healthy and diseased plants infected with Xf. Data are means of five replicates. Asterisks indicate statistical significance at 5% Tukey between diseased and healthy plants for each amino acid. ASP, aspartic acid; Glu, glutamic acid; Asn, asparagine; Ser, serine; Gln, glutamine; Arg, arginine.
30
20
10
0
Healthy Plant
Diseased Plant
Fig. 4. Amino acids contents (A, B) and proline content (C) in the xylem sap (A) and leaves (B, C) of healthy and diseased plants infected with Xf. Bars indicate standard deviation of five replicates. Different small letters indicate statistical significance at 5% Tukey between diseased and healthy plants.
mechanism controlling absorption by the roots, transport in the xylem and assimilation in the leaves. However, an increase of NO 3 in the leaves was not followed by an increase of NR as the enzyme activity did
not differ between DP and HP plants. This result might be explained by the complex control of NR activity in plants. The regulation of NR activity occurs initially by an effective control of the gene expression, determining the levels of the protein in the cytoplasm, with further control by reversible phosphorylation mechanisms (Kaiser et al., 1999). Both controls seem to be strongly influenced by cellular and environmental factors. NO 3 , light and CO2 concentration are among the main factors affecting NR level in the cells. The water stress symptom imposed by the CVC disease in sweet orange trees has been associated with alterations in the leaf gas exchange, as the CO2 concentration and water vapour in the leaves are dependent on the photosynthetic photon flux density (PPFD), vapour pressure
Nitrogen metabolism in citrus infected with Xylella fastidiosa 2739
deficit (VPD), and stomatal aperture, all being affected by the tissue water content (Medina, 2002; Gomes et al., 2004). The gas exchange in plants infected by Xf for 20 months and 26 months showed that CO2 assimilation was severely reduced, mainly in the first hours of the day and
Fig. 6. 2D-electrophoresis gels of proteins from the xylem sap of diseased (A, C) and healthy plants (B, D) stained with Coomassie Blue (C, D). The arrows indicate the proteins identified by mass spectrometry. DP01 to DP04 indicate the position of spots recovered from the Coomassie Blue stained gel from diseased plants while HP01 and HP02 are from the healthy plants. Protein molecular weight markers were phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), lysozyme (14.4 kDa), aprotinin (6.5).
at midday (Gomes et al., 2004). Therefore, the lack of a corresponding increased activity of NR, in response to a higher NO 3 concentration in the leaves of DP, may reflect a decrease in the internal CO2 concentration. Nicotiana plumbaginifolia plants, which had the posttranscriptional regulation of NR abolished, showed that CO2 controls the activity of the enzyme but not its expression, as the enzyme catalytic activity was maintained when these plants were kept in an atmosphere free of CO2, while normal plants showed a 60% decrease (Lejay et al., 1997). In addition, as reported in maize (Abd-El-Baki et al., 2000), a saline stress that develops in the DP might have affected the NR activity. Sodium was analysed in the leaves of the same plants used in the present study and this showed that DP accumulated 260 p.p.m., almost twice the content found in HP of 150 ppm (Purcino, 2006). Therefore, the accumulation of NO 3 in the leaves and xylem sap of DP was probably a consequence of a non-corresponding activity of NR in the roots as well in the leaves. On the other hand, GS activity, the enzyme responsible for the assimilation of inorganic NHþ 4 in amino acids, was higher in DP. Since there was no reduction of NO 3 by NR, as the levels of this nutrient were higher in DP, one may presume that the increased GS activity was a consequence of NHþ 4 reassimilation in photorespiration or by protein proteolysis. There are two isoforms of GS in plants which are differently regulated. GS1 is a cytosolic enzyme and GS2
Table 1. Identified proteins from 2D gel electrophoresis carried out with xylem sap collected from diseased (DP) and healthy (HP) plants Spot ID
Sequenced peptides
Best hits
HP01
QALKSCK
ABC transporter permease protein-like protein (Oryza sativa)
HP02
DP02 DP03
DP04
PGGPDNVNLAR MVLLVNPK LNDCPAR TFPAEYYLDLVSWVCMLLK LLSN LSPLTLCGNKR SVLLDGSDSEK SSDKLLYSSDEAESTTK EGKCSGVVSNVGSCRLAAR VATV GFEVLDALK GPIQLSWNYNYLR KASSDY YDSSAAGLP FFEDLLEYR QFFEDLLEYR EVKASSDYRKP GVPMSVTAV GSDGNVLACK TLDVPSPWK EYSYAYETR QGVNAVCR RSCPKAYSYAYDDR CAAFNEPKYCCTKFNKLEPFA
Mucin-like protein (Arabidopsis thaliana) Strictosidine synthase (Arabidopsis thaliana) Peroxidase precursor (Arabidopsis thaliana)
Chitinase (Citrus sinensis) Chitinase (Citrus jambhiri)
Thaumatin/PR5-like protein (Pyrus pyrifolia) Thaumatin-like protein (Prunus persica)
2740 Purcino et al.
Fig. 7. Summary of the results obtained and proposed mechanisms controlling the nitrogen metabolism in diseased plants. Up and down arrows indicate increase or decrease of concentration or activity. AA, amino acids, PRO, proline, ARG, arginine.
is plastidial (Lam et al., 1996). The use of mutants showed that GS2 is responsible for the assimilation of the NHþ 4 resulting from NO3 reduction or reassimilation from photorespiration (Blackwell et al., 1990), while GS1 is responsible for the reassimilation of the NHþ 4 resulting from protein degradation mainly during tissue senescence (Sakakibara et al., 1992). Infection by Xf increases the photorespiration in sweet orange leaves (Habermann et al., 2003; Ribeiro et al., 2003). Gas exchange and chlorophyll fluorescence measured in healthy and diseased plants showed a marked decrease of photosynthesis, but the photochemical quenching was similar (Ribeiro et al., 2003). However, when the measurements were carried out with an O2 electrode with 5% CO2, the photochemical quenching of the diseased plants showed a much larger decrease. This indicates that under normal atmospheric conditions (0.036% CO2) photorespiration was responsible for most of the quenching in these plants, as photosynthesis was lower than in healthy plants. Sweet orange trees with CVC symptoms present an intense chlorosis in the leaves, indicating that senescence might be taking place. Proteolysis is also a typical sign of senescence (Huffaker, 1990). Accordingly, DP leaves showed a marked increase of protease activity and a decrease of protein content. In the genome of Xf several sequences for proteases were found (Simpson et al., 2000) and the excretion of proteases by Xf in vitro was confirmed by a proteomic study, where a Zn-protease and two serine-proteases were identified (Smolka et al., 2003). Therefore, part of the increase of GS might be due to an increase in the release
of NHþ 4 by protease activity, although significant levels of NHþ 4 in HP and DP could not be detected. The excretion of proteases by Xf and their probable action on the cell proteins was suggested as nutritionally important as it might release peptides as well as being important for the radial migration of the bacteria through the xylem pits, since the Xf diameter is usually greater than the pore diameter (Simpson et al., 2000; Smolka et al., 2003). Recently, it was verified that an isolate of Xf from citrus secretes three major protease bands in SDS-PAGE activity gels containing gelatin as a copolymerized substrate (Fedatto et al., 2006). The protease activity was completely inhibited by PMSF and partially inhibited by EDTA indicating that proteases produced by Xf from sweet orange to the serine- and metallo-protease group, respectively (Fedatto et al., 2006) which is in agreement with proteomic studies (Smolka et al., 2003). Despite the higher protease activity it is not possible to determine if this is due to the excretion of proteases by Xf. However, these results clearly indicate, along with the chlorosis in the leaves, that a senescing process might be taking place in infected plants, which is partially supported by the lower protein content. On the other hand, the lower protein content in the sap may indicate that proteins released from the cell wall by the action of proteases are being used by the bacteria. In this regard, the bacterial biofilm may act as an exchanging support, as has been suggested for ions (Silva et al., 2001), retaining protein for bacterial use. A decrease of protein content as a consequence of an increased protease activity might have some effect on the concentration of amino acids, increasing their levels in the leaves of DP. The opposite was observed, but this result is in agreement with the results on the reduction of NO 3 and NHþ assimilation. One may assume that the amino acid 4 pool in DP leaves is more influenced by nitrogen- NO 3 assimilation than nitrogen recycling from proteolysis. On the other hand, the amino acid content was higher in the sap of DP, while Pro content in the leaves was similar in DP and HP plants. The participation of amino acids, particularly Pro (Delauney and Verma, 1993), as compatible solutes to reduce the water potential in water and salt-stressed plants has been an evidence of their importance in osmotic adjustment (Ogawa and Yamauchi, 2006). In this regard the results in the leaves and xylem sap of DP are very different. It is speculated that the higher content in the sap is a concentrating effect since, as indicated in the Materials and methods, sap bleeding was very low in DP. The expressive low concentration of amino acid in the leaves of DP, which during the hot period of the day showed wilting symptoms, suggests that osmotic adjustment did not occur in the leaves of DP. Osmotic adjustment was not observed in the leaves of lemon (C. limon) subjected to water stress (Ruiz-Sa´nchez et al.,
Nitrogen metabolism in citrus infected with Xylella fastidiosa 2741
1997) although it seems to occur in the fruits mainly because of sugar accumulation (Barry et al., 2004). These results showed that Arg was significantly higher in the sap and leaves of DP. The few publications on the amino acids in the xylem sap and leaves of sweet orange trees showed that Asn, Gln, and Arg are the main amino acids (Culian˜ez et al., 1981; Ramamurthy and Lu¨dders, 1982; Tadeo et al., 1984). They also reported that the composition is influenced by biotic stresses and environmental variations. The alterations in the amino acid composition in the xylem sap of P. persica infected by Xf could not be correlated with the presence of the bacteria (Gould et al., 1991). As observed here, Arg also increased in the leaves of sweet orange trees in the initial stages of Xf infection and at the same time subjected to water stress (Medina, 2002). However, no such increase was detected in infected plants that were regularly watered, leading to the conclusion that Arg accumulated as a response to water stress rather than Xf infection. Therefore, since plants were evaluated that had been infected by Xf for several months and showed visible symptoms of wilt during the hot periods of the day, such increases in Arg would be expected. At the same time, there was a lack of significant differences for Pro, a typical amino acid related to water stress (Hsiao, 1973; Hare and Cress, 1997). Pro in plants is synthesized in a well-established pathway derived from glutamate, however, there is increasing evidence that it may also be synthesized from the Arg biosynthesis pathway. In this case ornithine can be reversibly converted by ornithine-a-aminotransferase to a-ketod-aminovalerate, which is spontaneously converted to D1pyrroline-5-carboxylate (P2C) which, in turn, is reduced to Pro by P2C reductase (Coruzzi and Last, 2000). Therefore, the increase in Arg and the low, but not significant increase of Pro, may be a preferred biosynthesis towards Arg. The high content of Arg in DP might explain the levels of putrescine in these plants. Putrescine in plants can be synthesized from Arg by two routes, involving either ornithine or agmatine, although the decarboxylase for Arg (agmatine route) is more active than ornithine decarboxylase (Crozier et al., 2000; Martin-Tanguy, 2001). Several reports associate the formation of polyamines with different kinds of stresses (Walters, 2003). Putrescine was detected in appreciable amounts in the leaves and petioles of DP. As far as is known, there is no previous information on the polyamine content in citrus leaves although in the flavedo of Fortune mandarin (C. reticulata) putrescine is the main polyamine, whose concentration (approximately 250–300 lg g1 fresh weight) is 50 times higher than spermine and spermidine (Gonzalez-Aguilar et al., 1997, 1998). The immediate question emerging from this increased level of putrescine concerns its relationship with the disease. Is it a plant response to the Xf infection or is it
related to the water stress imposed by the xylem occlusion? Both situations, water stress and pathogens, are known to induce polyamines in plants. In some plants it has been suggested that the increase of polyamines in water-stressed Vicia faba occurs because they can control the stomatal aperture by modulating K+ channels in the membrane of the guard cells (Liu et al., 2000). In addition, the enhanced level of polyamines in some water-stressed plants has been associated with a protective mechanism against oxidative damage (Nayyar and Chander, 2004). Regarding their relationship in the complex interactions between pathogens and plants, several reports suggest that there is no general mechanism for plants, mainly when compatible and incompatible interactions are considered (Walters, 2000, 2003). Therefore, since it is not possible to dissociate CVC from water stress, it is not possible to conclude whether the variations observed for Arg and putrescine are related to any single situation. Perhaps both are involved, as arginine decarboxylase genes are expressed under different stressing situations (Mo and Pua, 2002). Proteome comparison of xylem sap
Marked differences were observed in the pattern of protein profile in 2D electrophoresis gels of saps from DP and HP. DP showed more low mass proteins which seems to be in agreement with the higher protease activity in these plants. The five spots successfully sequenced and identified did not have similarities with Xf sequences and were similar to mucin, strictosidine synthase, ABCtransporter protein, peroxidase, chitinase, and thaumatin. Permeases are proteins belonging to the family of ‘ATPbinding cassette (ABC) transporters and their involvement in plant–pathogen interactions has been suggested, as they are induced by ethylene, jasmonates, and salicylic acid (Campbell et al., 2003). Peroxidases belong to a large group of enzymes with a wide range of functions in plants (Welinder, 1992), including a clear relationship with resistance to pathogens (Wu et al., 1997; Zhou et al., 2002). Secreted by the epithelial cells of animal respiratory systems, the glycoprotein mucin interacts with cilia to retain pathogens and irritating inhaled substances (Li et al., 1998). Some reports have found nucleotide sequences with some degree of homology to mucin. As a chemical characteristic, glycoproteins can aggregate bacteria (Berrocal-Lobo et al., 2002) and, although not yet proved, it has been suggested that they might be involved in the resistance to insects (Wang et al., 2003). Strictosidine synthase is a key enzyme in the biosynthetic pathway of many indole alkaloids of Catharanthus roseus and other plants (Kutchan, 1993). Cell suspensions of C. roseus showed that strictosidine synthase was co-ordinately induced by fungi elicitors and jasmonate (Menke et al., 1999).
2742 Purcino et al.
Thaumatin belongs to the family PR5 of the pathogenesis-related proteins and besides their association with pathogens resistance they have also been suggested to be associated with senescence (Sassa et al., 2002). Some reports produced evidence that plant proteins displaying similarity with thaumatin have the ability to bind b-1, 3-glucans, conferring an anti-fungal activity (Trudel et al., 1998; Eulgem et al., 2004). All the five proteins identified have, in some way, a relationship with the response of plants to pathogens or stresses, which is a typical situation in citrus infected with Xf. In addition, the plants used in this work are not resistant to Xf and, therefore, it may only be concluded that the proteins sequenced from DP xylem sap do not confer resistance. However, 2D gels obtained in this study showed a variation in the protein profile and it is not possible to exclude that several of them are secreted by Xf, as has been observed in in vitro studies where proteases were investigated (Smolka et al., 2003; Fedatto et al., 2006).
Conclusion The results obtained in this study on several N compounds showed that Xf caused a marked disturbance in the N metabolism in citrus plants. All of them seem to be closely related, such as the variation of protein content and proteases, Arg and polyamines, and so on. However, since the bacteria clearly cause water stress in the plant it is not possible precisely to determine whether the microorganism or the stress caused all the observed variations. However, in some cases it seems that Xf benefits from this situation as the N availability seems to increase its growth. The detection of increased levels of protease activity show that radial migration may occur by the enlargement of pit pores in the xylem vessels. A strong limitation in the study of Xf in plants is that the development of the disease is slow. Therefore, colonization by the bacteria can only be certified after the pathogen has already spread to the plant tissues and important plant responses have occurred. The tissue preferentially colonized by Xf is the xylem and, in citrus, bacteria concentrate more in the petiole. This is also a limitation since any attempt to monitor disease development is destructive and the xylem is formed by dead cells. Since CVC was established as an important citrus disease, one strategy suggested as a means of controlling the disease is to study and understand the nutritional requirements of the bacteria since it inhabits one of the poorest nutritional environments in the plants. Acknowledgements This work received finantial support from Fundac xa˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP – project 01/03460-5).
RPP thanks FAPESP for a doctorate fellowship and PM, JCN, and MAM thank the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq-Brazil) for research fellowships. We also thank Dr Se´rgio Lilla (Galeno Research Unit, Campinas, SP, Brazil) for protein sequencing and Dr Helve´cio Della Coletta Filho for providing the primers for Xf detection by RT-PCR.
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