Peroxidase Activity Against Guaiacol, NADH, Chlorogenic Acid, Ferulic Acid and Coniferyl Alcohol in Root Tips of Lotus Japonicus and L

Biologia 65/2: 279—283, 2010 Section Botany DOI: 10.2478/s11756-010-0029-3

Peroxidase activity against guaiacol, NADH, chlorogenic acid, ferulic acid and coniferyl alcohol in root tips of Lotus japonicus and L. corniculatus grown under low pH and aluminium stress

Veronika Zelinová1, Igor Mistrík1, Peter Paľove-Balang1,2, Ladislav Tamás 1*

1Institute of Botany, Slovak Academy of Sciences, Dúbravská cesta 14,SK-84523 Bratislava, Slovakia; e-mail: [email protected] 2Department of Botany, Faculty of Natural Sciences, University of P. J. Šafárik, Mánesova 23,SK-04154 Košice, Slovakia

Abstract: The purpose of this study was to examine the impact of low pH and Al stress on the apoplastic production of H2O2 and POD activity against guaiacol, ferulic acid, coniferyl alcohol, NADH and chlorogenic acid in the root tip (RT) of two cultivars of Lotus corniculatus and the model Lotus japonicus Gifu, with the goal to determine the possible role of POD activity in proton and Al tolerance. Our results suggest that Lotus corniculatus cv. UFRGS is more tolerant to low pH and Al than cv. Draco due to the high POD activities involved in CW strengthening. The enzymatic response of Lotus japonicus Gifu is similar rather to the sensitive cultivar. On the other hand, in cvs Draco and Gifu low pH induced the activation of CW-modifying PODs, which is probably a component of the defence response of roots to the presence of toxic protons. Aluminium did not activate further these activities suggesting that defence response and acclimation to low pH confers also effective defence against Al toxicity in Lotus species. The activity of NADH-POD and CGA-POD were not affected significantly by exposure to low pH or Al. Al and pH induced drop in H2O2 production, which was more relevant in cv. Gifu and Draco than in cv. UFRGS is probably associated with enhanced activity of peroxidases involved in secondary CW metabolism utilizing H2O2 as an electron acceptor. Key words: chlorogenic acid peroxidase; coniferyl alcohol peroxidase; ferulic acid peroxidase; guaiacol peroxidase; NADH peroxidase Abbreviations: CW, cell wall; CGA-POD, chlorogenic acid peroxidase; CoA-POD, coniferyl alcohol peroxidase; FA-POD, ferulic acid peroxidase; G-POD, guaiacol peroxidase; HM, heavy metal; NADH-POD, NADH peroxidase; POD, peroxidase; ROS, reactive oxygen species; RT, root tip

Introduction et al. 1997). In acidic solutions (pH < 5.0), Al exist 3+ as the octahedral hexahydrate, Al(H2O)6 ,whichby Aluminium (Al) is the most abundant metal in the convention is usually called Al3+(Kochian 1995). This Earth’s crust, existing mainly as oxides and aluminosil- form of Al appears to be the most important rhizotoxic icates, which are harmless to organisms. However, these Al species, which interferes with a wide range of cel- biologically inactive insoluble Al forms are solubilized in lular processes (Delhaize & Ryan 1995; Kochian 1995; acid conditions and released as phytotoxic species into Matsumoto 2000). Plant cell wall (CW) is the first tar- the soil. Due to the acidification of environments, owing get site of both proton and Al action. The distribu- to several anthropogenic factors, the area of acid soils tion of absorbed Al is different among plant species but (comprises approximately 30% of the world’s ice-free major part of absorbed Al (30–90%) was found in the land area) is gradually increased from year to year (von apoplast (Rengel 1996). Al rapidly interferes with the Uexküll & Mutert 1995). Therefore, low soil pH (pro- CW structure, where Al strongly binds to the negative ton toxicity) and Al toxicity became the major growth- charges of CW pectic matrix affecting its permeability limiting factors for crop cultivation in several countries and extensibility. Therefore, the modification of CW throughout the world. may lead either to decrease or to increase of Al tol- It has been observed that some varieties of given erance. Schmohl & Horst (2000) unequivocally proved plant species are proton tolerant but Al sensitive or that NaCl adapted cells with high pectin content bind vice versa (Gunsé et al. 1997). On the other hand it more Al, and are more sensitive to Al than control cells has been also demonstrated that some plants adapted containing lower amount of pectin in their CW. In addi- to low soil pH are mostly tolerant to Al (Watanabe tion, already low pH causes irreversible damage to the

* Corresponding author

c 2010 Institute of Botany, Slovak Academy of Sciences 280 V. Zelinová et al. growing Arabidopsis root by the disturbance of pectin- compared with the Lotus japonicus cv. Gifu, a model Ca interaction (Koyama et al. 2001). Gunsé et al. (1997) legume which is genetically closely related to Lotus cor- have reported that maize root CW were less elastic in niculatus. acid stressed than in control seedlings. In wheat, Al markedly induced deposition of lignin in the elonga- Material and methods tion region of roots mainly in the Al-sensitive genotypes (Sasaki et al. 1996). Most recently it has been reported Plant material and growth conditions that CW polysaccharides play an important role in ex- The 5 cm long shoot cuttings of Lotus japonicus cv. Gifu cluding Al from rice root apex (Yang et al. 2008). B-129 and Lotus corniculatus cvs. INIA-Draco and UFRGS (seeds were obtained from Dr. Monica Rebuffo – INIA La In plants, CW modifications are generally observed Estanzuela, Colonia, Uruguay) were planted in vermiculite. processes during both abiotic and biotic stress re- Plants were grown in controlled conditions (20 ◦C, 50% rel- sponses, where a crucial role belongs to peroxidases ative humidity, 16h/8h photoperiod and approximately 150 −2 −2 (PODs, EC 1.11.1.7; hydrogen donor: H2O2 oxido- µmol m s illumination) on “Hornum” nutrient solution reductase). These heme-containing glycoproteins using described by Handberg & Stougaard (1992). After 10 days hydrogen peroxide as an electron acceptor and several the plants were washed and transferred to 3 L containers for substrates as electron donors are involved in a wide the establishment of hydroponics. The hydroponic medium range of physiological processes (Hiraga et al. 2001). consisted of 1/10 strength Hornum media with some mod- CW crosslinking may be catalyzed by POD through ifications (1 mM CaCl2,0.5mMKNO3,0.5mMNH4NO3 and 0.5 mM KCl). The medium was continuously aerated, the oxidative coupling of feruloyl-polysaccharides (Fry replaced every 2 days. After 8 days, the plants were trans- 1986). POD activities involved in lignification are com- ferred to new solutions, containing 1 mM CaCl2,0.5mM monly analyzed against the coniferyl alcohol as a sub- KNO3,0.5mMNH4NO3 and 0.5 mM KCl and treated with strate (Boudet et al. 1995). Besides these classical per- 0.5 or 2 mM AlCl3 for 5 days. The pH was maintained at pH oxidative reactions some isoperoxidases in the pres- 4.0 or 5.5 (±0.2) by continuous monitoring with pH meter ence of Mn2+ ions and phenolic compounds, and using supplemented with Autoburette system (Radiometer TTT NADH as a substrate can participate in the produc- 80 and ABU 80, Copenhagen, Denmark) using 10 mM HCl. tion of ROS (Schopfer et al. 2001; Liszkay et al. 2003). In addition, it has also been proposed that PODs can Protein extraction and enzyme assays Root tips (1 cm) were excised into cold 50 mM Tris-HCl function in H2O2 scavenging using e.g. chlorogenic acid buffer, pH 8.0 containing 1 M NaCl and homogenized using as a substrate (Takahama & Oniki 1997). cold pestle and mortar. The homogenate was centrifuged The Leguminosae (the family Fabaceae) are sec- at 12000×g for 15 min. The proteins were quantified with ond only to the Gramineae in importance to humans as bovine serum albumin as the calibration standard by the a source of food, feed for livestock and raw materials method of Bradford (1976). for industry (Graham & Vance 2003). Apart from their Guaiacol (G-POD) activity was measured in a reac- use as food and feed, legumes are used in soil conser- tion mixture containing 50 mM Na-acetate buffer (pH 5.2), vation, phytoremediation, lumber production, as orna- 0.04% guaiacol and 0.04% H2O2 and 0.5 µgofproteins.The mental herbs and bushes, and for extraction of gums, absorbance was measured with the microplate reader at 470 nm over 1 min at 30 ◦C. Using ferulic acid as a substrate resins or food additives (Udvardi et al. 2005). Numer- the reaction mixture contained 50 mM Na-phosphate buffer ous abiotic and biotic impediments limit yield potential (pH 6.0), 100 µM ferulic acid and 0.02% H2O2 and 0.5 µg in legumes, including: drought, soil salinity, soil acidity of proteins. Enzyme activity (FA-POD) was expressed as ◦ and nutrient limitations, and various diseases and pests ∆A310 after incubation for 1 min at 30 C. Coniferyl alco- (Graham & Vance 2003). Among legumes, forage Lotus hol POD (CoA-POD) activity was determined in a reaction species have greater potential for adaptation to envi- mixture containing 50 mM K-phosphate buffer (pH 7.0), 0.3 ronmentally constrained areas. The main Lotus species mM coniferyl alcohol and 0.2 mM H2O2 and 0.5 µgofpro- teins. Enzyme activity was expressed as ∆A262 after incuba- with high forage value are L. corniculatus, L. glaber, L. ◦ subbiflorus and L. uliginosus. Abiotic stresses, such pe- tion for 1 min at 30 C. The H2O2 generating POD activity (NADH-POD) was measured using NADH as a substrate. riodic drought, soils salinity and soil acidity are among The reaction mixture contained 50 mM Tris-Maleate buffer the most important environmental constrains to Lotus pH 7.0, 1 mM 4–aminoantipyrine; 1 mM 2,4–dichlorophenol; productivity in the region of the South America and 50 mM MnCl2; and 0.2 mM NADH and 1 µgofproteins. negatively determine livestock production. Developing The increase in absorbance was measured at 510 nm af- plants that are tolerant to these stresses remain an im- ter incubation for 5min at 30 ◦C. Using chlorogenic acid as portant aim of breeding programmes and enhance pro- a substrate the reaction mixture contained 100 mM Na- ductivity and sustainability of pastures in the Southern phosphate buffer pH 6.0, 0.1 mM chlorogenic acid and 1 mM H2O2 and 0.25 µg of proteins. The enzyme activity Cone of Latin America. ◦ The purpose of the study reported in this paper wasexpressedas∆A320 after incubation for 1 min at 30 C. was to examine the apoplastic production of H2O2 and Hydrogen peroxide assay POD activity against guaiacol, ferulic acid, coniferyl Hydrogen peroxide (H2O2 ) production was monitored fluo- alcohol, NADH and chlorogenic acid in the root tip rometrically using the Amplex Red Hydrogen Peroxide As- (RT) in two cultivars of Lotus corniculatus differing in say Kit (Molecular Probes) according to manufacturer’s rec- stress tolerance to evaluate their possible role in proton ommendations with minor modifications. Root tips (1 cm and Al tolerance. The response of these cultivars was long, 15 per reaction) were incubated in 200 µLofthe50 Peroxidase activity in roots of Lotus species grown under low pH and Al 281

pH 5.5 pH 4.0 0.5 mM Al 2 mM Al pH 5.5 pH 4.0 0.5 mM Al 2 mM Al 1.800 0.700 1.600 * * 0.600 1.400 1.200 * 0.500

* A 310 nm] 1.000 * ∆ 0.400 0.800 0.300 0.600 0.400 0.200

G - POD activity [A 470 nm] activity - POD G 0.200 0.100 0.000 FA - POD activity [ Gifu Draco UFRGS 0.000 Gifu Draco UFRGS Fig. 1. Guaiacol-POD activity in the apical part of root at pH 5.5 (control), pH 4.0, and at 0.5 or 2 mM Al 5 days after exposure Fig. 2. Ferulic acid-POD activity in the apical part of root at pH of Lotus seedlings. Data represent the means ± SD (n =5).*– 5.5 (control), pH 4.0, and at 0.5 or 2 mM Al 5 days after exposure values significantly different for P ≤ 0.05. of Lotus seedlings. Data represent the means ± SD (n =5).*– values significantly different for P ≤ 0.05. mM sodium phosphate buffer, pH 7.4 containing 25 µMAm- plex Red reagent (from 10 mM DMSO stock solution) and stress responses (Ishii 1997). The formation of difer- ◦ 0.02 U of horse radish peroxidase for 30 min at 30 C. Flu- ulic acid by the oxidation of ferulic acids ester-linked orescence signal was recorded (150 µL of reaction mixture to the CW polysaccharides leads to the crosslinking of without root segments) with the microplate reader (Synergy matrix polysaccharides that causes CW rigidification HT BIO-TEK, USA) using excitation at 530 (filter 530/25) (Kamisaka et al. 1990). In our experiments increased nm and fluorescence detection at 590 (filter 590/20) nm. FA-POD activity was observed only in L. japonicus Statistical analyses cv. Gifu during acid and Al exposure (Fig. 2). How- Results represent the mean of five independent experi- ever both cvs of L. corniculatus have had a high FA- ments. The significance of differences between control and POD activity already in control roots in comparison Al-treatments was analysed using Student’s t-test. to L. japonicus seedlings. Similarly to FA-POD activity, the lignification associated CoA-POD activity was Results and discussion the lowest in the roots of L. japonicus under control conditions and its activity increased both by proton The genera Lotus is considered to be more tolerant to or Al toxicity with exception of cv. UFRGS (Fig. 3). acidic soils then most of other legumes and have greater UFRGS contains high CoA-POD activity already at potential for adaptation to environmentally constrained control conditions (pH 5.5). In wheat, Al treatment in- areas (Edmeades et al. 1991; Correa et al. 2001). Lotus creased the contents of ferulic and p-coumaric acid espe- japonicus cv. Gifu has been characterized as moderate cially in the Al-sensitive cultivar by increasing activity tolerant to both acidity and Al, while Lotus cornicula- of phenylalanine ammonia lyase (Hossain et al. 2006). tus cv. Draco as drought tolerant, and cv. UFRGS as Tabuchi & Matsumoto (2001) have suggested that Al Al tolerant (Paľove-Balang & Mistrík 2007; Pavlovkin makes the CW thick and rigid, thereby inhibiting the et al. 2009). root growth through the elevated amount of ferulic and Plants exposed to stress either abiotic or biotic diferulic acids in the CW. High activation of lignifying generally showed up-regulated peroxidase activity (Pas- enzymes during drought may be a injurious symptom, sardi et al. 2005). In barley, the Al-induced root growth which leads to reduced growth of seedlings (Lee et al. inhibition and the enhancement of Al-induced peroxi- 2007). On the other hand, spatially localized changes dase activity were positively correlated and were more in CW modification such as crosslinking or lignification activated in Al-sensitive cultivar than in Al-tolerant one are involved in the inhibition of root growth but may fa- (Tamás et al. 2003). Similar results were obtained in cilitate root acclimation to drying or toxic environment our experiments with Lotus species, where a higher Al- (Fan et al. 2006). CW lignification and suberization of induced G-POD activity was detected in cvs Gifu and epidermis and exodermis in the RT of Phragmites aus- Draco than in a more tolerant UFRGS (Fig. 1). The tralis are interpreted as a defence reaction that limits acid environment did not activate G-POD activity ex- the entry of toxic metals into the roots (Ederli et al. ception of the drought tolerant cv. Draco where a slight 2004). It has been reported that the two times higher but significant increase was observed. lignin deposition was detected into the root endoder- Guaiacol is the most commonly used non-specific mis in hyperaccumulator Thlaspi caerulescens in com- artificial substrate for POD activity assay, and its ele- parison to Arabidopsis thaliana (van de Mortel et al. vated activity may indicate a stress condition for plants. 2006). Similarly in Matricaria chamomilla roots, rapid By using of more specific substrates we can analyze accumulation of phenolic compounds and lignin con- some specific role of different peroxidases during these tent were observed as early as one day after Cu or stress responses. In plants, CW modifications are gener- Cd treatment (Kováčik & Klejdus 2008). It has been ally observed processes during both abiotic and biotic suggested that strengthening of the CW may protect 282 V. Zelinová et al.

pH 5.5 pH 4.0 0.5 mM Al 2 mM Al pH 5.5 pH 4.0 0.5 mM Al 2 mM Al 1.400 0.800 * 1.200 0.700 * * 1.000 0.600 A 320 nm] A 262 nm] ∆ ∆ 0.500 0.800 0.400 0.600 0.300 0.400 0.200 0.200 0.100 CoA - POD activity [ CGA - POD activity [ 0.000 0.000 Gifu Draco UFRGS Gifu Draco UFRGS

Fig. 3. Coniferyl alcohol-POD activity in the apical part of root Fig. 5. Chlorogenic acid-POD activity in the apical part of root at pH 5.5 (control), pH 4.0, and at 0.5 or 2 mM Al 5 days after at pH 5.5 (control), pH 4.0, and at 0.5 or 2 mM Al 5 days after exposure of Lotus seedlings. Data represent the means ± SD (n = exposure of Lotus seedlings. Data represent the means ± SD (n = 5). * – values significantly different for P ≤ 0.05. 5). * – values significantly different for P ≤ 0.05.

pH 5.5 pH 4.0 0.5 mM Al 2 mM Al pH 5.5 pH 4.0 0.5 mM Al 2 mM Al 0.600 9.000

0.500 8.000 7.000 0.400 6.000 0.300 5.000 0.200 4.000

0.100 3.000 NADH - POD activity [A 510 nm] activity NADH - POD 0.000 2.000 Gifu Draco UFRGS 1.000

Fig. 4. NADH-POD activity in the apical part of root at pH 5.5 0.000 (control), pH 4.0, and at 0.5 or 2 mM Al 5 days after exposure Gifu Draco UFRGS of Lotus seedlings. Data represent the means ± SD (n =5).*– values significantly different for P ≤ 0.05. Fig. 6. Production of H2O2 intheapicalpartofrootatpH5.5 (control), pH 4.0, and at 0.5 or 2 mM Al 5 days after exposure of Lotus seedlings. Data represent the means ± SD (n =5).*– plants from metal toxicity through the prevention from values significantly different for P ≤ 0.05. efflux of metals from xylem vessels (van de Mortel et al. 2008), but on the other hand caused CW thicken- (Fig. 4 and 5) suggesting that in these Lotus cvs the ing and rigidification, thereby the inhibition of root generation of ROS is not activated during Al stress. growth. It is probably that the cv. UFRGS just due Similarly to FA-POD and CoA-POD the highest CGA- to its high POD activity associated with CW strength- POD activity during control conditions was detected ening is more tolerant to Al than other two analyzed in cv. UFRGS. This high CGA-POD activity probably Lotus cvs, however in which their activation is occurred. confers also to enhanced tolerance to oxidative stress. Another possible mechanism is that more rigid and lig- In addition we detected a decrease in H2O2 production nified CW is a protection against Al by reducing its ca- which was more relevant in cvs Gifu and Draco than pability to bind Al and therefore becomes more tolerant in cv. UFRGS (Fig. 6). Decreased H2O2 level may be than an unmodified CW. This hypothesis is supported associated with an enhanced activity of peroxidases in- also by observation that the exposure of Arabidopsis to volved in secondary CW metabolism utilizing H2O2 as low pH stress caused lethal injuries in growing tissues an electron acceptor. while non-growing tissues remained viable (Koyama et In conclusion, our results suggest that L. cornic- al. 2001). Similarly to low pH, it has been reported that ulatus cv. UFRGS due to the high POD activities in- the most Al sensitive part of root tip is the distal part of volved in CW strengthening is more tolerant both to transition zone (Sivaguru & Horst 1998). These results low pH and Al than cv. Draco and the response of the suggest that secondary modified CW is more tolerant to model L. japonicus Gifu is similar rather to the sen- proton or Al toxicity than primary CW. The protection sitive cultivar. On the other hand, in cvs Draco and of CW from Al binding is crucial tolerant mechanism, Gifu low pH induced the activation of CW-modifying which has been demonstrated in several plant species PODs, which is probably a component of the defence (Li et al. 2009). response of roots to the presence of toxic protons. Al The activity of NADH-POD as a ROS generating did not activate these activities further, suggesting that POD and CGA-POD as a ROS scavenging POD were defence response and acclimation to low pH confers also not affected significantly by exposure to low pH or Al effective defence against Al toxicity in Lotus species. Peroxidase activity in roots of Lotus species grown under low pH and Al 283

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