Xylella fastidiosa disturbs nitrogen metabolism and causes a stress response in sweet orange Citrus...

Journal of Experimental Botany, Page 1 of 12

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RESEARCH PAPER

Xylella fastidiosa disturbs nitrogen metabolism and causesa stress response in sweet orange Citrus sinensis cv. Pera

Rubia P. Purcino1,2, Camilo Lazaro Medina3, Daniel Martins4, Flavia 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, Brazil2 Universidade Federal Rural do Rio de Janeiro, Km 7 Rodovia BR 465, 23850-000, Seropedica, RJ, Brazil3 Conplant, Rua Francisco Andreo Aledo, 22, 13084-200, Campinas, SP, Brazil4 Departamento de Bioquımica, Instituto de Biologia, Universidade Estadual de Campinas, CP 6109, 13083-970,Campinas, SP, Brazil5 Instituto Agronomico de Campinas, Centro de Ecofisiologia e Biofısica, Av. Barao de Itapura 1481, CP 28,13012-970, Campinas, SP, Brazil6 Instituto Agronomico de Campinas, Centro APTA Citros Sylvio Moreira, CP 04, 13490-970, Cordeiropolis,SP, Brazil

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.

‘Pera’ (C. sinensis L. Osbeck) infected with Xf. Plants

were artificially infected and maintained in the green-

house until they have developed clear disease symp-

toms. 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 causingseveral 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: pmazza@unicamp.br

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/) whichpermits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Xylella fastidiosa has caused important economic lossesin the Brazilian citrus industry where it is known as citrusvariegated chlorosis (CVC), first detected in Brazil in themid-1980s (Rossetti et al., 1990) and since then hasspread to all citrus-growing areas in Brazil and SouthAmerica (Carvalho et al., 1995). The economical impor-tance of CVC has led to complete genome sequencing ofXf (Simpson et al., 2000).It has been suggested that the primary mechanism of

pathogenicity of Xf is occlusion of the xylem vessels bythe biofilm formed by the bacteria (Souza et al., 2006).This mechanism seems to explain satisfactorily most ofthe symptoms shown by infected plants, however, bio-synthesis of toxins and alterations in hormonal levels mayalso be involved in the pathogenicity of Xf (Hayward andMariano, 1997).Several genes coding for proteases were found in the Xf

genome (Simpson et al., 2000) as well as one Zn-proteaseand two serino-proteases that were identified in a proteo-mic analysis of proteins excreted by Xf cultivated in vitro(Smolka et al., 2003). Proteases might facilitate the lateralmovement of the bacteria in the xylem vessels, bydigesting protein of the cell wall and increasing the pitdiameter (Simpson et al., 2000). In this process, enzymescapable of degrading the cell wall carbohydrates mightalso be involved in pit enlargement (Simpson et al.,2000). However, proteases might have a nutritionalimportance for Xf growth in the xylem by releasing smallpeptides 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 compoundssuch as amino acids, organic acids, and inorganicnutrients, they are usually found in low concentrations,limiting bacterial growth (Purcell and Hopkins, 1996).Glutamine (Gln) and asparagine (Asn) are the main aminoacids in the xylem sap of plants (Lea et al., 2007) and forthis reason they have been included in several artificialmedia for Xf growth (Davis et al., 1980; Chang andDonaldson, 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 isstill under dispute.Very little is known about the nitrogen (N) composition

of the Citrus xylem sap. Asn and Gln appear to be themain amino acids in the sap transported from the roots tothe shoot of Satsuma mandarins (C. unshiu) (Tadeo et al.,1984). However, the amino acid composition and contentseem to vary in the xylem sap of citrus according to theperiod of the year (Culianez et al., 1981; Ramamurthy andLudders, 1982; Tadeo et al., 1984). On the other hand,stresses caused by diseases (young-tree decline) seem toenhance the content of amino acids in citrus leaves,particularly arginine (Arg) and praline (Pro). However, norelationship 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.

Pera grafted on Rangpur lime (C. limonia) in the initialstages of CVC development (Medina, 2002). The increaseof Arg content in leaves of sweet orange under stress hasbeen suggested as a detoxification process, where thedecrease of glucose content due to photosynthesis limita-tion cause an accumulation of NHþ

4 to toxic levels (Rabeand Lovatt, 1986). Such a relationship between Argbiosynthesis and NHþ

4 accumulation has been reported byother authors (Lovatt, 1986; Sagee and Lovatt, 1991).However, low levels of NHþ

4 were detected in leaves ofsweet orange Pera and no variation was observed inwater-stressed or non-stressed plants whether infected ornot with Xf (Medina, 2002).To our knowledge, there is no information in the

literature on the enzymes of N metabolism in sweetorange infected by Xf. The NO�

3 transported in the xylemvessels to the leaves is sequentially reduced to nitrite andNHþ

4 by the enzymes nitrate reductase (NR) and nitritereductase, respectively (Lea et al., 1990). Then NHþ

4 - Nwill be incorporated by glutamine synthetase (GS, EC6.3.1.2) into glutamate to form Gln using NHþ

4 andglutamate as substrates (Lea et al., 1990). Much of theGln is then used for the biosynthesis of other amino acids,mainly Asn, which can also be transported in the xylemsap (Lea et al., 1990).The aim of this investigation was to study N

metabolism in sweet orange ‘Pera infected with Xf, byanalysing nitrogen compounds and activities of pro-teases, NR, and GS in the xylem sap and leaves ofhealthy (HP) and diseased plants (DP). In addition,proteins extracts from the petiole of HP and DPwere separated by 2D-PAGE electrophoresis aiming toreveal differentially expressed proteins in HP and DPand their relationship with N metabolism and stressresponse.

Materials and methods

Plant material

The variety sweet orange ‘Pera’, which is highly susceptible to Xf,was grafted onto ‘Rangpur’ lime (C. limonia Osbeck) and infectedwith Xf by grafting with infected budwoods (Nunes et al., 2004).Healthy and infected plants were kept in a greenhouse in100 l barrels containing a mixture of soil, manure and sand (3:1:1,by vol.). The greenhouse at the Centro de Citricultura SylvioMoreira, Instituto Agronomico de Campinas, Cordeiropolis (22�34’S; 47�34’ W; 689 m altitude), SP, Brazil, was completely closed toprevent the passage of insects. When these plants were approxi-mately 5-year-old they were used in the experiments. The plantswere watered by a drip irrigation system and fertilized regularly,according to the recommendations for citrus (Grupo Paulista deAdubacxao e Calagem para Citros, 1994). All sap and leaf sampleswere collected from 2–6 July 2004, from 08.00–10.00 h.

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Xylem sap collection

Sap was collected from the apical part (3–5 expanded leaves) ofbranches from DP and HP. Because of the obstruction of the xylemby the bacteria a pressure pump was used (Gould et al., 1991). Thebranches were removed with a razor blade and immediatelymounted in the pressure chamber. Then, the cut surface was washedwith distilled water, blotted dry with absorbent paper and the gasreleased to increase the pressure in the chamber. Once sap bleedingwas observed at the cut stem the pressure was increased up to 10%of the water potential observed at bleeding and then held for sapcollection. The sap of several plants was collected until a volume ofapproximately 1 ml was reached. Five 1 ml replicates were obtainedfor 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 transpirationis still reasonable in the DP (Medina, 2002). In the HP the sap wasabundant even at low pressures (3–5 bars) while for DP pressuresup to 15 bars were necessary. The withdrawn sap was collectedwith 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 inRT-PCRs (Missanvage et al., 1994; Pooler and Hartung, 1995). RT-PCR 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 1.5min (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 unitTaq 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 liquidN2, then extracted with cold (4 �C) 5 ml 0.1 M Na-phosphatebuffer, 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 wascentrifuged 20 min at 16 000 g at 4 �C and the supernatantrecovered 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 minwith 5 ml of 100 mM Na-phosphate buffer, pH 7.5, containing25 mM KNO3 and 1% isopropanol. The leaf discs were maintainedfor 30 min at 35 �C in the dark, under agitation, and then boiled for5 min. The enzyme activity was determined by the amount of NO2

released by the discs in the incubation media after boiling. Samplesof 2 ml were mixed 1:1 with 1% sulphanilamide in a mixture of2.4 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 powderedin liquid N2 and then proteases extracted with 100 mM TRIS–HCl,pH 7.5, followed by centrifugation. The reaction was carried outin Eppendorf tubes by initially mixing 50 ll leaf extract or xylemsap with 450 ll 25 mM TRIS–HCl, pH 7.5. After 10 minof preincubation at 40 �C, 200 ll 1% azocasein was added tothe reaction and incubation proceeded for 6 h at the sametemperature. Then, 350 ll was withdrawn from each tube and mixedwith 150 ll 10% trichloroacetic acid. After 20 min at roomtemperature the mixture was centrifuged and the absorbance of thesupernatant measured at 440 nm.

NO�3 , NH

þ4 , amino acids, soluble proteins, and polyamines

The same extracts obtained for GS activity were used to measuretotal soluble proteins (Bradford, 1976), NO�

3 (Cataldo et al., 1975),

NHþ4 (Sodek and Lea, 1993) and soluble amino acids (Cocking and

Yemm, 1954). In the sap, the measurements of these substanceswere made after centrifugation to eliminate the bacteria.Qualitative analyses of amino acids were carried out by HPLC

(Shimadzu) with fluorimetric detection after derivatization witho-phthalaldehyde (Jarret et al., 1986). A Spherisorb ODS-2C18 column was used with 0.8 ml min�1 flow rate for a lineargradient formed by solution A, 65% methanol and solution B, pH7.5 phosphate buffer (50 mM sodium acetate, 50 mM disodiumphosphate, 1.5 ml acetic acid, 20 ml tetrahydrofuran, and 20 mlmethanol in 1.0 l H2O). The gradient was the proportion of solutionA 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 and40 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 and61 min. The amino acids eluting from the column were monitoredby a Shimadzu fluorescence detector operating on a 250 nmexcitation wavelength and a 480 nm emission wavelength. Twentymicrolitres of the amino acid solution and 40 ll of the OPA solutionwere mixed and, after 2 min, 20 ll of the mixture were injected intothe HPLC.To measure proline in the sap the samples were derivatized with

the AccQ-Fluor Reagent Kit (Waters) following the manufacturer’sinstructions and then analysed by HPLC. In the leaves, Pro wasmeasured with ninhydrin (Ringel et al., 2003). Polyamines weredetected 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 coldacetone:ethanol:acetic acid solution (50:49.9:0.1 by vol.) at –20 �C,overnight. Precipitated proteins were dissolved in 50 ll of 10 mMTRIS (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 theresulting protein extracts was used to determine the proteinconcentration using a Bradford protein assay kit (Bio-Rad). Theprotocols for the 2D electrophoresis are as described in Smolkaet al. (2003). Analyses were carried out in triplicate.

Protein identification by mass spectrometry

Proteins were identified by mass spectrometry using Q-TOFequipment (Applied Biosystems). The protein spots were excisedfrom 2D gels and submitted to in-gel digestion for theextraction of peptides, according to Schevechenko et al. (1996).The mass spectra were obtained by using a hybrid Q-ToF massspectrometer (Q-ToF Ultima, Micromass, Manchester, UK) witha Zspray source operating in the positive mode. The ionizinationconditions include capilar voltage of 2.3 kV, cone and lensvoltage RF1 of 30 V and 100 V, respectively, and collisionenergy of 10 eV. The temperature of the source was –70 �C andthe nitrogen gas on the cone with the flux of 80 l h�1. Argon gaswas used to refrigerate the collision and fragmentation of ions inthe collision cell. The external calibration was done with sodiumiodide for a mass scale from 50 m/z to 3000 m/z. All spectra wereacquired with the ToF analyser in V mode (ToF kV¼9.1) andMCP voltage of 2150 V.By using the BLASTX tool (http://www.ncbi.nlm.nih.gov/

BLAST/) the peptide sequences obtained were compared withprotein sequences in public databanks (NCBI), using the proteindatabase of the Brazilian Xf proteome project (http://www.proteome.ibi.unicamp.br/index-xylella.htm), the translated nucleo-tide sequences of the CitEST database of Citrus expressed sequencetags (http://biotecnologia.centrodecitricultura.br/), and the translatedsequences of the Brazilian Xf genome project (http://aeg.lbi.ic.unicamp.br/xf/).

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Statistics

Data were analysed by ANOVA and means were compared by theTukey test at 5% significance.

Results

Confirmation of Xf infection

The yield of sap exudation in the plants infected with Xfwas very low because of the obstruction of the xylemvessels by the bacteria. Therefore, samples taken fromseveral plants were mixed until 1 ml was obtained. Inorder to verify the presence of Xf in these composedsamples, PCR assays were carried out using two sets ofprimers specific for the detection of Xf (Missanvage et al.,1994; Pooler and Hartung, 1995). Both primers were usedin the same reactions and therefore two bands ofamplification were expected in the sap from DP, as shownin Fig. 1. At the same time, no bands were observed in thesap from HP.

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 thexylem sap of DP (Fig. 2A, B). However, using colorimet-ric and HPLC methods only traces of NHþ

4 could bedetected in leaves but no positive results in the sapsamples.Despite the higher concentration of NO�

3 in DP, theactivity of NR did not differ between DP and HP plants(Fig. 3A). On the other hand, GS activity was significantlyhigher in DP (Fig. 3B).Besides a reduction in the protein content in the xylem

sap and leaves (Fig. 3C, D), DP also showed a markedincreased level of protease activity in the xylem sap andleaves (Fig. 3E, F).Although not statistically significant, there was a clear

trend for a lower content of amino acids in the leaves ofDP (Fig. 4). On the other hand, the amino acid contentwas higher in the sap of the same plants (Fig. 4A). Proline(Pro) content in the leaves was similar in DP and HPplants (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 thexylem saps (Fig. 5A). Other amino acids were in very lowconcentration and are not shown. Significant differenceswere observed for Asn and Arg. Asn showed the highestdecrease from 40.3 mol% in HP to 25.7 mol% in the DP.By contrast, Arg showed an increase in DP, from12.7 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 acidsdetected were Asp, Glu, Asn, Ser, Gln, Arg, and Ala, and

the others were below 5 mol% and are not shown. Themost contrasting differences were found in Gln and Arg(Fig. 5B). While Arg concentration was higher in DP, Glnconcentration was lower in these plants.HPLC analysis showed that only putrescine was found

in appreciable amounts in the tissues of DP whoseconcentrations were 15.3 lg g�1 and 37.7 lg g�1 freshweight in the petiole and leaves, respectively. Polyamines

Fig. 1. RT-PCR reactions for the detection of Xf in the xylem sapcollected from healthy and diseased plants. Markers are shown on bp ¼base pairs.

Healthy Plant Diseased Plant

0

40

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120

160

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2,0A leaves

B xylem sap

NO

3

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g.g

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w

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ht)

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3

1- (µg

.m

L-1 xylem

sap

)

a

a

a

b

Fig. 2. Nitrate concentration in the leaves and xylem sap of healthyand diseased plants infected with Xf. Bars indicate standard deviation offive replicates. Different small letters indicate statistical significance at5% Tukey between diseased and healthy plants.

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were undetectable in the leaves of HP while traces(1.32 lg g�1 fresh weight) were detected in the petioles.

Proteome comparison of xylem sap

The patterns of protein profile in 2D electrophoresis gelsstained with silver nitrate are shown in Fig. 6A and B.Besides differences in the intensity of several spots, it maybe observed that DP has more low mass proteins andfewer high molecular mass proteins. Even when doublingthe amount of proteins loaded on the gels, many fewerprotein spots could be observed with CB staining, butsome of the proteins which showed a marked difference interms of spot intensity remained and six were selected forsequencing (Fig. 6C, D). Five spots were successfullysequenced and identified but they did not have similaritieswith Xf sequences. These peptides were similar to mucin,strictosidine synthase, ABC-transporter protein, peroxi-dase, chitinase, and thaumatin (Table 1).

Discussion

NO�3 , enzyme activities, proteins, amino acids, and

polyamines

Figure 7 is a scheme summarizing all the results obtainedin this study and is intended to be a guide in thediscussion.NO�

3 accumulated in the leaves and in the xylem sap ofDP, and only a trace of NHþ

4 was detected in the leavesbut not in the sap samples. The higher concentration ofNO�

3 in the DP might be a consequence of an alteredN metabolism in the roots, with reduced incorporation inamino acids. The transport of NO�

3 from the roots to otherparts of the plants depends on the NO�

3 assimilation whichdepends on the NO�

3 concentration in the soil, a processhighly controlled by a feedback mechanism related to theaccumulation of amino acids, mainly Gln (Gojon et al.,1998). Therefore, higher NO�

3 accumulation in DP mightbe a consequence of alterations in this complex

0

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100

120

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Healthy Plant Diseased Plant Healthy Plant Diseased Plant

Healthy Plant Diseased Plant

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Healthy Plant Diseased Plant

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Pro

tease (ab

s x 100)

E xylem sap F leaves

0

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40

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120

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tease (ab

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14D leaves

a aa

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Fig. 3. Activities of the enzymes nitrate reductase (A), glutamine synthetase (B), and protein (C, D) contents and protease activity (E, F) in thexylem sap (C, E) and leaves (D, F) of healthy and diseased plants infected with Xf. Bars indicate standard deviation of five replicates. Different smallletters indicate statistical significance at 5% Tukey between diseased and healthy plants.

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mechanism controlling absorption by the roots, transportin the xylem and assimilation in the leaves.However, an increase of NO�

3 in the leaves was notfollowed by an increase of NR as the enzyme activity did

not differ between DP and HP plants. This result might beexplained by the complex control of NR activity in plants.The regulation of NR activity occurs initially by aneffective control of the gene expression, determining thelevels of the protein in the cytoplasm, with further controlby reversible phosphorylation mechanisms (Kaiser et al.,1999). Both controls seem to be strongly influenced bycellular and environmental factors. NO�

3 , light and CO2

concentration are among the main factors affecting NRlevel in the cells.The water stress symptom imposed by the CVC disease

in sweet orange trees has been associated with alterationsin the leaf gas exchange, as the CO2 concentration andwater vapour in the leaves are dependent on the photo-synthetic photon flux density (PPFD), vapour pressure

A xylem sap

0

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min

o acid

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(η

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Healthy Plant Diseased Plant

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30

40

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lin

e (µ

mo

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C

a

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a

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aa

Fig. 4. Amino acids contents (A, B) and proline content (C) in thexylem sap (A) and leaves (B, C) of healthy and diseased plants infectedwith Xf. Bars indicate standard deviation of five replicates. Different

0

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Asp Glu Asn Ser Gln Arg

Am

in

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s (m

ol%

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0

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60B leaves

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Healthy plantA

min

o acid

s (m

ol%

)

*

*

*

*

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 fivereplicates. Asterisks indicate statistical significance at 5% Tukeybetween diseased and healthy plants for each amino acid. ASP, asparticacid; Glu, glutamic acid; Asn, asparagine; Ser, serine; Gln, glutamine;Arg, arginine.

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deficit (VPD), and stomatal aperture, all being affected bythe tissue water content (Medina, 2002; Gomes et al.,2004). The gas exchange in plants infected by Xf for 20months and 26 months showed that CO2 assimilation wasseverely reduced, mainly in the first hours of the day and

at midday (Gomes et al., 2004). Therefore, the lack ofa corresponding increased activity of NR, in response toa higher NO�

3 concentration in the leaves of DP, mayreflect a decrease in the internal CO2 concentration.Nicotiana plumbaginifolia plants, which had the post-transcriptional regulation of NR abolished, showed thatCO2 controls the activity of the enzyme but not itsexpression, as the enzyme catalytic activity was main-tained when these plants were kept in an atmosphere freeof 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 inthe DP might have affected the NR activity. Sodium wasanalysed in the leaves of the same plants used in thepresent study and this showed that DP accumulated260 p.p.m., almost twice the content found in HP of150 ppm (Purcino, 2006). Therefore, the accumulation ofNO�

3 in the leaves and xylem sap of DP was probablya consequence of a non-corresponding activity of NR inthe roots as well in the leaves.On the other hand, GS activity, the enzyme responsible

for the assimilation of inorganic NHþ4 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, onemay presume that the increased GS activity was a conse-quence of NHþ

4 reassimilation in photorespiration or byprotein proteolysis.There are two isoforms of GS in plants which are

differently regulated. GS1 is a cytosolic enzyme and GS2

Fig. 6. 2D-electrophoresis gels of proteins from the xylem sap ofdiseased (A, C) and healthy plants (B, D) stained with Coomassie Blue(C, D). The arrows indicate the proteins identified by mass spectrom-etry. DP01 to DP04 indicate the position of spots recovered from theCoomassie Blue stained gel from diseased plants while HP01 and HP02are from the healthy plants. Protein molecular weight markers werephosphorylase 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).

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)

PGGPDNVNLARMVLLVNPK Mucin-like protein (Arabidopsis thaliana)LNDCPAR Strictosidine synthase (Arabidopsis thaliana)TFPAEYYLDLVSWVCMLLK

HP02 LLSN Peroxidase precursor (Arabidopsis thaliana)LSPLTLCGNKRSVLLDGSDSEKSSDKLLYSSDEAESTTKEGKCSGVVSNVGSCRLAARVATVGFEVLDALK

DP02 GPIQLSWNYNYLR Chitinase (Citrus sinensis)KASSDYYDSSAAGLP

DP03 FFEDLLEYR Chitinase (Citrus jambhiri)QFFEDLLEYREVKASSDYRKPGVPMSVTAV

DP04 GSDGNVLACK Thaumatin/PR5-like protein (Pyrus pyrifolia)TLDVPSPWK Thaumatin-like protein (Prunus persica)EYSYAYETRQGVNAVCRRSCPKAYSYAYDDRCAAFNEPKYCCTKFNKLEPFA

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is plastidial (Lam et al., 1996). The use of mutantsshowed that GS2 is responsible for the assimilation of theNHþ

4 resulting from NO�3 reduction or reassimilation from

photorespiration (Blackwell et al., 1990), while GS1 isresponsible for the reassimilation of the NHþ

4 resultingfrom 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 mea-sured in healthy and diseased plants showed a markeddecrease of photosynthesis, but the photochemicalquenching was similar (Ribeiro et al., 2003). However,when the measurements were carried out with an O2

electrode with 5% CO2, the photochemical quenching ofthe diseased plants showed a much larger decrease. Thisindicates that under normal atmospheric conditions(0.036% CO2) photorespiration was responsible for mostof the quenching in these plants, as photosynthesis waslower than in healthy plants.Sweet orange trees with CVC symptoms present an

intense chlorosis in the leaves, indicating that senescencemight be taking place. Proteolysis is also a typical sign ofsenescence (Huffaker, 1990). Accordingly, DP leavesshowed a marked increase of protease activity anda decrease of protein content.In the genome of Xf several sequences for proteases

were found (Simpson et al., 2000) and the excretion ofproteases by Xf in vitro was confirmed by a proteomicstudy, where a Zn-protease and two serine-proteases wereidentified (Smolka et al., 2003). Therefore, part of theincrease of GS might be due to an increase in the release

of NHþ4 by protease activity, although significant levels of

NHþ4 in HP and DP could not be detected.

The excretion of proteases by Xf and their probableaction on the cell proteins was suggested as nutritionallyimportant as it might release peptides as well as beingimportant for the radial migration of the bacteria throughthe xylem pits, since the Xf diameter is usually greaterthan the pore diameter (Simpson et al., 2000; Smolkaet al., 2003). Recently, it was verified that an isolate of Xffrom citrus secretes three major protease bands inSDS-PAGE activity gels containing gelatin as a copolym-erized substrate (Fedatto et al., 2006). The proteaseactivity was completely inhibited by PMSF and partiallyinhibited by EDTA indicating that proteases produced byXf from sweet orange to the serine- and metallo-proteasegroup, respectively (Fedatto et al., 2006) which is inagreement 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 proteasesby Xf. However, these results clearly indicate, along withthe chlorosis in the leaves, that a senescing process mightbe taking place in infected plants, which is partiallysupported by the lower protein content. On the other hand,the lower protein content in the sap may indicate thatproteins released from the cell wall by the action ofproteases are being used by the bacteria. In this regard, thebacterial biofilm may act as an exchanging support, as hasbeen suggested for ions (Silva et al., 2001), retainingprotein for bacterial use.A decrease of protein content as a consequence of an

increased protease activity might have some effect on theconcentration of amino acids, increasing their levels in theleaves of DP. The opposite was observed, but this result isin agreement with the results on the reduction of NO�

3 andNHþ

4 assimilation. One may assume that the amino acidpool in DP leaves is more influenced by nitrogen- NO�

3

assimilation than nitrogen recycling from proteolysis. Onthe other hand, the amino acid content was higher in thesap of DP, while Pro content in the leaves was similar inDP and HP plants.The participation of amino acids, particularly Pro

(Delauney and Verma, 1993), as compatible solutes toreduce the water potential in water and salt-stressed plantshas been an evidence of their importance in osmoticadjustment (Ogawa and Yamauchi, 2006). In this regardthe results in the leaves and xylem sap of DP are verydifferent. It is speculated that the higher content in the sapis a concentrating effect since, as indicated in theMaterials 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 dayshowed wilting symptoms, suggests that osmotic adjust-ment did not occur in the leaves of DP. Osmoticadjustment was not observed in the leaves of lemon (C.limon) subjected to water stress (Ruiz-Sanchez et al.,

Fig. 7. Summary of the results obtained and proposed mechanismscontrolling the nitrogen metabolism in diseased plants. Up and downarrows indicate increase or decrease of concentration or activity. AA,amino acids, PRO, proline, ARG, arginine.

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1997) although it seems to occur in the fruits mainlybecause 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 theamino acids in the xylem sap and leaves of sweet orangetrees showed that Asn, Gln, and Arg are the main aminoacids (Culianez et al., 1981; Ramamurthy and Ludders,1982; Tadeo et al., 1984). They also reported that thecomposition is influenced by biotic stresses and environ-mental variations. The alterations in the amino acidcomposition in the xylem sap of P. persica infected byXf could not be correlated with the presence of thebacteria (Gould et al., 1991).As observed here, Arg also increased in the leaves of

sweet orange trees in the initial stages of Xf infection andat the same time subjected to water stress (Medina, 2002).However, no such increase was detected in infected plantsthat were regularly watered, leading to the conclusionthat Arg accumulated as a response to water stress ratherthan Xf infection. Therefore, since plants were evaluatedthat had been infected by Xf for several months andshowed visible symptoms of wilt during the hot periods ofthe day, such increases in Arg would be expected. At thesame time, there was a lack of significant differences forPro, 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 increasingevidence that it may also be synthesized from the Argbiosynthesis pathway. In this case ornithine can be reversiblyconverted by ornithine-a-aminotransferase to a-keto-d-aminovalerate, which is spontaneously converted to D1-pyrroline-5-carboxylate (P2C) which, in turn, is reduced toPro by P2C reductase (Coruzzi and Last, 2000). Therefore,the increase in Arg and the low, but not significant increaseof 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 besynthesized from Arg by two routes, involving eitherornithine or agmatine, although the decarboxylase for Arg(agmatine route) is more active than ornithine decarbox-ylase (Crozier et al., 2000; Martin-Tanguy, 2001). Severalreports associate the formation of polyamines withdifferent 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 noprevious information on the polyamine content in citrusleaves although in the flavedo of Fortune mandarin (C.reticulata) putrescine is the main polyamine, whoseconcentration (approximately 250–300 lg g�1 freshweight) 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 thedisease. Is it a plant response to the Xf infection or is it

related to the water stress imposed by the xylemocclusion? Both situations, water stress and pathogens,are known to induce polyamines in plants. In some plantsit has been suggested that the increase of polyamines inwater-stressed Vicia faba occurs because they can controlthe stomatal aperture by modulating K+ channels in themembrane of the guard cells (Liu et al., 2000). Inaddition, the enhanced level of polyamines in somewater-stressed plants has been associated with a protectivemechanism against oxidative damage (Nayyar andChander, 2004). Regarding their relationship in thecomplex interactions between pathogens and plants,several reports suggest that there is no general mechanismfor plants, mainly when compatible and incompatibleinteractions are considered (Walters, 2000, 2003).Therefore, since it is not possible to dissociate CVC

from water stress, it is not possible to conclude whetherthe variations observed for Arg and putrescine are relatedto any single situation. Perhaps both are involved, asarginine decarboxylase genes are expressed under differ-ent stressing situations (Mo and Pua, 2002).

Proteome comparison of xylem sap

Marked differences were observed in the pattern ofprotein profile in 2D electrophoresis gels of saps from DPand HP. DP showed more low mass proteins which seemsto be in agreement with the higher protease activity inthese plants. The five spots successfully sequenced andidentified did not have similarities with Xf sequences andwere similar to mucin, strictosidine synthase, ABC-transporter protein, peroxidase, chitinase, and thaumatin.Permeases are proteins belonging to the family of ‘ATP-

binding cassette (ABC) transporters and their involvementin plant–pathogen interactions has been suggested, as theyare 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 toretain pathogens and irritating inhaled substances(Li et al., 1998). Some reports have found nucleotidesequences with some degree of homology to mucin. Asa chemical characteristic, glycoproteins can aggregatebacteria (Berrocal-Lobo et al., 2002) and, although notyet proved, it has been suggested that they might beinvolved in the resistance to insects (Wang et al., 2003).Strictosidine synthase is a key enzyme in the biosyn-

thetic pathway of many indole alkaloids of Catharanthusroseus and other plants (Kutchan, 1993). Cell suspensionsof C. roseus showed that strictosidine synthase wasco-ordinately induced by fungi elicitors and jasmonate(Menke et al., 1999).

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Thaumatin belongs to the family PR5 of the pathogen-esis-related proteins and besides their association withpathogens resistance they have also been suggested to beassociated with senescence (Sassa et al., 2002). Somereports produced evidence that plant proteins displayingsimilarity 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 orstresses, which is a typical situation in citrus infected withXf. In addition, the plants used in this work are notresistant to Xf and, therefore, it may only be concluded thatthe proteins sequenced from DP xylem sap do not conferresistance. However, 2D gels obtained in this study showeda variation in the protein profile and it is not possible toexclude that several of them are secreted by Xf, as has beenobserved in in vitro studies where proteases wereinvestigated (Smolka et al., 2003; Fedatto et al., 2006).

Conclusion

The results obtained in this study on several N compoundsshowed that Xf caused a marked disturbance in theN metabolism in citrus plants. All of them seem to beclosely related, such as the variation of protein contentand proteases, Arg and polyamines, and so on. However,since the bacteria clearly cause water stress in the plant itis not possible precisely to determine whether the micro-organism or the stress caused all the observed variations.However, in some cases it seems that Xf benefits from thissituation as the N availability seems to increase its growth.The detection of increased levels of protease activity showthat radial migration may occur by the enlargement of pitpores 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 thepathogen has already spread to the plant tissues andimportant plant responses have occurred. The tissuepreferentially colonized by Xf is the xylem and, in citrus,bacteria concentrate more in the petiole. This is alsoa limitation since any attempt to monitor disease de-velopment is destructive and the xylem is formed by deadcells. Since CVC was established as an important citrusdisease, one strategy suggested as a means of controllingthe disease is to study and understand the nutritionalrequirements of the bacteria since it inhabits one of thepoorest nutritional environments in the plants.

Acknowledgements

This work received finantial support from Fundacxao de Amparo aPesquisa do Estado de Sao Paulo (FAPESP – project 01/03460-5).

RPP thanks FAPESP for a doctorate fellowship and PM, JCN, andMAM thank the Conselho Nacional de Desenvolvimento Cientıficoe Tecnologico (CNPq-Brazil) for research fellowships. We alsothank Dr Sergio Lilla (Galeno Research Unit, Campinas, SP, Brazil)for protein sequencing and Dr Helvecio Della Coletta Filho forproviding the primers for Xf detection by RT-PCR.

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