Biochemical response of grapevine variety ‘Chardonnay’ (Vitis vinifera L.) to infection with grapevine yellows (Bois noir)

Biochemical response of grapevine variety ‘Chardonnay’(Vitis vinifera L.) to infection with grapevine yellows(Bois noir)

Denis Rusjan & Heidi Halbwirth & Karl Stich &

Maja Mikulič-Petkovšek & Robert Veberič

Accepted: 2 April 2012 /Published online: 17 April 2012# KNPV 2012

Abstract This study was carried out on the leaves ofphytoplasma susceptible grapevine variety ‘Chardonnay’(Vitis vinifera L.), and included research of the alterationsin chlorophyll and carotenoid contents, contents of phe-nol compounds and in related enzymes activity in thephenylpropanoid pathway during the Bois noir (BN)infection. Phytoplasma-infected leaves showed reducedcontents of chlorophylls and carotenoids, which promot-ed their susceptibility to oxidative reactions. Furthermore,modulation of flavonoid biosynthesis occurred in infectedleaves, leading to an increased activity of phenylalanineammonia lyase, chalcone synthase/chalcone isomerase,flavanone 3-hydroxylase and polyphenoloxidase, but to adecreased peroxidase activity. Phytoplasma infection ledto an increase of the contents of hydroxycinnamic acids(caftaric acid, sinapic acid glucose derivate and coutaricacid), flavanols (procyanidin B1, procyanidin dimer 3,catechin, epicatechin) and flavonols (quercetin 3-O-glucuronide, quercetin 3-O-glucoside) especially in theperiod up to vérasion. The study demonstrated that at

certain phenological key-stages infection with phyto-plasma (BN) induced different alterations in enzymeactivities and in the contents of biochemical compoundsfrom primary and secondary metabolism.

Keywords Bois noir . Carotenoids . Chlorophyll .

Oxidases . Phenylalanine ammonia lyase .

Phenylpropanoid pathway . Phytoplasma

Grapevine yellows diseases, especially Bois noir (BN),are serious phytosanitary problems not only in Europebut nowadays all over the world. BN is caused byaccidentally transmitted infection with phytoplasmabelonging to the stolbur group (16SrXII-A subgroup)(Maixner et al. 1995; Sforza et al. 1998). Grapevine is anaccidental host of phytoplasma and responses to itsinfection with various symptoms and metabolic altera-tions such as inhibition of the biosynthesis of chloro-phyll and carotenoids and an accumulation of certaingroups of phenolic compounds (Mikulič-Petkovšek etal. 2007; Slatnar et al. 2010), has an impact on theplants’ natural antioxidative defence mechanism againstphytoplasma infections (Bertamini et al. 2003). Inhibi-tion of photosynthetic activity reduces the accumulationof soluble carbohydrates (sucrose) and starch in theleaves (Maust et al. 2003) of the infected plants. Thisleads to a modification of secondary metabolism, espe-cially of phenylpropanoid biosynthesis (Ferri et al.2011). Grapevine polyphenols have been the subject ofnumerous studies during the past decade, especially

Eur J Plant Pathol (2012) 134:231–237DOI 10.1007/s10658-012-9988-2

D. Rusjan (*) :M. Mikulič-Petkovšek : R. VeberičBiotechnical Faculty, Agronomy Department,University of Ljubljana,Jamnikarjeva 101,1000 Ljubljana, Sloveniae-mail: [email protected]

H. Halbwirth :K. StichTechnical University of Vienna,Institute for Chemical Engineering,Getreidemarkt 9/1665,1060 Vienna, Austria

during infection by fungi, where the inhibition mecha-nisms of fungal enzymes involved in pathogenesis wasmediated by tannins and other phenolic compounds(Mahoney et al. 2003).

Phenylalanine ammonia lyase (PAL; E.C.4.3.1.24) isthe key enzyme at the interface between primary andsecondary metabolism and contributes to several path-ways including phenylpropanoid biosynthesis. Further-more, Archana et al. (2011) suggested that theconstitutive upregulation of phenylalanine ammonia-lyase is one of the main factors responsible for protec-tion against pathogen attack. Chalcone synthase (CHS;E.C.3.2.1.74) and chalcone isomerase (CHI;E.C.3.2.1.14) are required for the formation of the firstreal flavonoid structures in the pathway, the flavanones,which are further converted by flavanone 3-hydroxylase(FHT; E.C. 1.14.11.9) into dihydroflavonols, which arethe substrates for dihydroflavonol 4-reductase (DFR;E.C.1.1.1.219), a key enzyme in the biosynthesis ofsynthesis of the anthocyanins, proanthocyanidins, andflavonols (Pourcel et al. 2007), the last of which areknown to play the scavenger of reactive oxygen species.Furthermore, Pourcel et al. (2007) reported the complexmode ac t ion o f po lypheno l ox idases PPO(E.C.1.14.18.1) and peroxidase (POD; E.C.1.11.1.7),which is regulated at several levels, involving transcrip-tional to post-translational mechanisms and the differ-ential subcellular compartmentalization of enzymes andsubstrates. A positive correlation between the levels ofpolyphenol oxidases (PPO; E.C.1.14.18.1) and theresistance to pathogens was demonstrated (Pourcel et al.(2007), whereas the role of peroxidase (POD;E.C.1.11.1.7) in the living plant is still poorly understood.

The aim of this study was to investigate the biochem-ical response of the highly sensitive grapevine variety tophytoplasma (BN) infections at different phenologicalkey-stages, regarding the enzyme activities of the phenyl-propanoid pathway, polyphenols and pigment contents inleaves, to study the specific impact and enrich scientificknowledge of phytoplasma (BN) phytopathology.

The study was carried out on the virus-free 20-yearsold vines planted in the vineyard, Slovenia (ELISAtest at vérasion; GFLV, GLRaV-1, -2, -3, -6 and −7,GVA and GFkV showing negative results at all tests;Boscia et al. 1997). Leaves were sampled from fiveinfected (INF) and five uninfected (HLT) vines at fourdifferent phenological key-stages: 20th July (BBCH75; berries pea-size; early stage when symptoms ofinfection on leaves were not yet visible), 2nd August

(BBCH 81; vérasion; phytoplasma-infected symptom-atic leaves visible), 9th September (BBCH 89; harvest;phytoplasma-infected symptomatic leaves visible) and1st October (BBCH 92; leaf colouring; leaves fromuninfected plants also discoloured but due to seasonalchanges). The infection with phytoplasma BN wasconfirmed by PCR with specific primer on DNAwhich was extracted using DNeasy Plant Mini kits(Qiagen, Hilden, Germany).

Extraction of chlorophyll and carotenoids fromfresh foliage was carried out with dimethyl sulfoxide(DMSO). Concentrations of single photosynthetic pig-ments (chlorophyll a and b, and carotenoids) in theextracts were determined following Wellburn (1994).The total phenolic content (TPC) of the extracts wasassessed using the Folin-Ciocalteu phenol reagentmethod and expressed as gallic acid equivalents(GAE) in mg per g FW of leaf.

Phenol compounds from leaves were extractedaccording to the method reported by Mikulič-Petkovšeket al. (2007). Analysis was carried out using MS2 scan-ning from m/z 115 to 1100. Concentrations of phenoliccompounds were calculated from the peak areas of thesamples and the corresponding standards and expressedin mg kg−1 fresh weight (FW).

PAL, CHS/CHI and FHT extractions were conductedaccording to the method described by Thill (2010),whereas those for POD and PPO followed Goessingeret al. (2009). The enzyme assays were adapted fromHalbwirth et al. (2002) for grapevine leaf (Table 1). Toremove low molecular compounds, 400 μl of superna-tant was passed through a gel chromatography column(Sephadex G25medium). The protein content was quan-tified by a modified Lowry procedure (Sandermann andStrominger 1972) with crystalline BSA as standard. PPOand POD activity was carried out according to the Wor-thington manual (Worthington Biochemical Corporation1972). Enzyme activity was calculated in nkat g−1 FWfor PAL, CHS/CHI and FHT, and in ΔA min−1 for PPOand POD.

The average concentration of chlorophyll a, chloro-phyll b and carotenoids are outlined in Table 2. Theinfection with BN significantly decreased the averagecontents of chlorophylls by 28–65 % and of carotenoidsby 23–57 % during all vegetation stages-a strongereffect than reported earlier by Gutha et al. (2010) forvirus-infected leaves. Furthermore, the infection withBN did not affect the Chl a/Chl b ratio, which was stableduring the entire vegetation period, what coincided with

232 Eur J Plant Pathol (2012) 134:231–237

the results of González et al. (1997) for viral infection.On the other hand, the obtained results partly contradictthe results of Blanchfield et al. (2006) and Petit et al.(2006) especially regarding carotenoids.

Irrespective of infection, the total phenols content inleaves decreased nearly throughout the whole vegeta-tion period; however at the last sampling the statisticallyhighest content was determined in uninfected leaves(Fig. 1). The opposite impact on the total phenol content

in the infected leaves at the last sampling can beexplained by faster degradation of phenolic compoundsin leave tissues, due to the lower contents of protectivemetabolites (pigments; Table 2), possible biodegrada-tion caused by different microorganisms (Alexander1965), and after flow disruption of primary metabolites,especially of carbohydrates (Ferri et al. 2011).

The infection with BN statistically increased thecontents of hydroxycinnamic acids in leaves, but only

Table 1 Optimised conditions regarding substrates, cofactors, buffers and their volumes used for assay of studied enzymes involved inthe phenylpropanoid pathway in leaves of grapevine ‘Chardonnay’

Enzyme Abbreviation Substrates μl Cofactor andco-substratesolutions

μl Assay buffer Buffer(ml)

Finalvolume(μL)

Phenylalanine ammonialyase

PAL (14C)-phenylalanine(0.063 nmol, 548 Bq)

5 0.1 M H3BO3 * 55 100pH 8.5

Chalcone synthase/chalcone isomerase

CHS/CHI p-CuCoA (1 nmol)(14C)-Malonyl-CoA(1.5 nmol, 1300 Bq)

5 0.1 M KH2PO4

*50 100

5 pH 8.0

Flavanone 3-hydroxylase FHT (14C)-naringenin(0.036 nmol, 100 Bq)

2-Oxoglutarate(1.46 mg mL−1)

5 0.1 M Tris/HCl*

60 100

FeSO4 x 7 H2O(1.46 mg mL−1)

5 pH 7.5

Polyphenol oxidase PPO Pyrocatechol (0.20 M) 170 0.1 M Na2HPO4

pH 6.5330 600

Peroxidase POD o-Dianisidine(0.410 nmol)

10 0.1 M KH2

PO4 **1050 1110

pH 6.5

* containing 0.4 % Na-ascorbate; ** containing 0.1 M H2O2

Table 2 Concentrations of chlorophyll a, chlorophyll b and carotenoids (mean±s.e.; n010) in leaves of grapevine ‘Chardonnay’according to BN-infection

Treatment Sampling

20th July 2nd Aug 9th Sept 1st Oct

Chlorophyll a (μg mg−1)

HLT 1.26±0.04 b 1.61±0.09 b 1.41±0.08 b 0.92±0.08 b

INF 0.91±0.06 a 1.07±0.15 a 0.49±0.03 a 0.37±0.02 a

Chlorophyll b (μg mg−1)

HLT 0.50±0.03 b 0.66±0.03 b 0.60±0.02 b 0.42±0.03 b

INF 0.36±0.02 a 0.49±0.05 a 0.21±0.01 a 0.16±0.02 a

Ratio (Chl a/Chl b)

HLT 2.52±0.07 2.34±0.11 2.32±0.05 2.17±0.10

INF 2.51±0.06 2.35±0.06 2.31±0.06 2.32±0.13

Carotenoids (μg mg−1)

HLT 91.1±3.8 b 111.2±5.0 b 102.4±5.1 b 79.2±5.5 b

INF 70.1±4.9 a 80.1±10.3 a 44.5±2.2 a 34.2±2.5 a

Values in a horizontal row followed by a different letter are significantly different at P<0.05 by LSD multiple range test

Eur J Plant Pathol (2012) 134:231–237 233

up to véraison, where the most abundant acids werecaftaric, sinapic acid glucose derivate and coutaricacid. After véraison the contents of hydroxycinnamicacids in leaves still decreased irrespective of infectionbut statistical differences between infected and unin-fected plants were not observed (Fig. 1).

The infection with BN also resulted in an additionalaccumulation of flavanols, which statistically differednearly throughout the whole vegetation period, exceptat the last sampling where the statistically highestcontent was determined in uninfected leaves (Fig. 1).At the early phenological stages the most abundantflavanols in infected leaves were procyanidin B1, pro-cyanidin dimer 3, catechin, and epicatechin whichpartly corresponds with the report of Treutter andFeucht (1990) and Gutha et al. (2010) for fungal-and viral-infection. A statistically significant increase

of flavonol contents was observed only at early vege-tation stages, where the contents of quercetin 3-O-glucuronide and quercetin 3-O-glucoside predomi-nated as shown for fungal infection (Koskimäki et al.2009). The phytoplasma-infected non-symptomaticleaves were abundant also in quercetin 3-O-galactoside,kaempferol 3-O-glucoside, and kaempferol 3-O-arabinoside. An alteration of flavonol contents inphytoplasma-infected symptomatic leaves was not ob-served in later vegetation stages.

PAL activity was strongly induced in phytoplasma-infected leaves throughout the whole vegetation period(Fig. 2). The CHS/CHI activity generally increasedduring the vegetation period and with the exception ofthe last sampling date, infected plants showed increasedCHS/CHI activity (Fig. 2). Higher sensitivity to phyto-plasma infection was observed for FHT activity, which

Fig. 1 Concentrations (mean±s.e.) of hydroxycinnamic acids, fla-vanols and flavonols in mg kg−1 of FW in leaves of ‘Chardonnay’according to the infection with phytoplasma (BN). Those columnswith a different lower-case letter at each sampling date denote

statistically significant differences by LSD multiple range test atP<0.05. ANOVAwas carried out independently for each samplingdate


was statistically highest in infected leaves during thewhole vegetation period and it peaked in latest vegeta-tion (Fig. 2).

Infection with phytoplasma statistically increased thePPO activity in leaves, especially in late phenologicalstages, which confirms the claim of Mayer and Harel(1979) that viral, bacterial or fungal challenge can inducePPO activity. There are many contradictory affirmations

about the role of POD in the context of pathogen infec-tion. Bindschedler et al. (2006) and Yusupova et al.(2006) reported that PODs play a significant role ingenerating H2O2 in the context of defence response andin conferring resistance to a wide range of pathogens.Moreover, Tománková et al. (2006) affirmed that thepathogen-resistant accession intensifies the productionof H2O2, thereby increasing the POD activity. However,

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Fig. 2 Enzyme activity [mean±s.e. (nkat g−1 FW) for phenyl-alanine ammonia lyase (PAL), chalcone synthase/chalconeisomerase (CHS/CHI) and flavanone 3-hydroxylase (FHT) andin ΔA min−1 for peroxidase (POD) and polyphenoloxidase(PPO)] in leaves of ‘Chardonnay’ according to infection with

phytoplasma, BN. Those columns with a different lower-caseletter at each sampling date denote statistically significant differ-ences by LSD multiple range test at P<0.05. ANOVA wascarried out independently for each sampling date


our results showed that the statistically higher PODactivities were present in uninfected leaves, especiallyin late vegetation stages (harvest and later).

The present study showed for the first time theeffect of phytoplasma-infection (BN) on flavonoidenzymes and related phenolic compounds in leavesof grapevine variety ‘Chardonnay’ at the main pheno-logical stages during vegetation. In context of primarymetabolism, it was clearly shown that the infectionwith BN decreased the contents of chlorophyll andcarotenoids in leaves. This could also affect the furtheractivities in secondary metabolism, especially regard-ing the hydroxycinnamic acids and flavonol synthesisat the beginning and the synthesis of flavanolsthroughout vegetation. The infection induced theactivity of many key enzymes (PAL, CHS/CHI, FHTand PPO), but not of POD, which showed differentdynamics. The results of the study showed that theimpact of BN infection on the contents of phenolcompounds and enzyme activities, involved in phenyl-propanoid pathway, are conditional upon phenologicalstage.

Acknowledgement This work is part of the programmeHorticulture No. P4-0013-0481 and the project No. J4-0890funded by the Slovenian Research Agency.

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Biochemical response of grapevine variety ‘Chardonnay’ (Vitis vinifera L.) to infection with grapevine yellows (Bois noir)