JXB Advance Access originally published online on July 10, 2006
Journal of Experimental Botany 2006 57(11):2825-2835; doi:10.1093/jxb/erl044
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RESEARCH PAPER |
Nematode infection and reproduction in transgenic and mutant Arabidopsis and tobacco with an altered phenylpropanoid metabolism
1Laboratory of Tropical Crop Improvement, Division of Crop Biotechnics, Catholic University of Leuven (K.U. Leuven), Kasteelpark Arenberg 13, 3001 Leuven, Belgium
2Unité de Chimie Analytique, Faculté Universitaire des Sciences Agronomiques de Gembloux, Passage des Déportés 2, 5030 Gembloux, Belgium
*To whom correspondence should be addressed. E-mail: dirk.dewaele{at}biw.kuleuven.be
Received 17 January 2006; Accepted 5 May 2006
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Abstract |
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Transgenic and mutant Arabidopsis and tobacco plants with altered phenylpropanoid metabolism were infected with the plant parasitic root knot nematode Meloidogyne incognita to assess the effect of the transgene or mutation on nematode infection and reproduction. Modifications in the lignin biosynthetic pathway which alter lignin composition in roots affected reproduction. In Arabidopsis with increased levels of syringyl lignin, reproduction was lower than in wild-type plants, while in tobacco with reduced levels of syringyl lignin, life cycle progression was stimulated. Overexpression of a MYB transcription factor of phenylpropanoid metabolism in tobacco significantly stimulated reproduction of M. incognita, while overexpression of L-phenylalanine ammonia-lyase had no effect. Arabidopsis transparent testa mutants with deficiencies in flavonoid pathway enzymes did not affect reproduction of M. incognita in the present infection tests.
Key words: Chlorogenic acid, flavonoid, guaiacyl lignin, Meloidogyne incognita, syringyl lignin, transparent testa mutants
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Introduction |
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The phenylpropanoid pathway of secondary metabolism has been altered by metabolic engineering and induced mutations mainly in Arabidopsis [Arabidopsis thaliana (L.) Heynh.] and tobacco (Nicotiana tabacum L.), with the purpose of increasing knowledge on pathway structure, regulation, and flux control. Engineering provides options for increased lignin degradation (paper industry) and forage digestibility, for the enhancement of the antioxidant capacity of food for health reasons, and for increased disease resistance. Pathway regulation and flux control have been investigated in tobacco by sense and antisense expression of L-phenylalanine ammonia-lyase (PAL) and cinnamic acid 4-hydroxylase (C4H), two enzymes of the ‘core’ phenylpropanoid pathway from which lignin and flavonoid pathways are derived (Howles et al., 1996; Blount et al., 2000). Jung et al. (2000) and Yu et al. (2000) transferred the gene coding for isoflavone synthase from soybean to non-leguminous species—Arabidopsis, tobacco, and maize—and obtained the synthesis of legume-specific isoflavonoids in these plants. With respect to alterations in lignin metabolism, different endogenous and heterologous enzyme-coding genes were expressed in sense and antisense direction mainly in tobacco but also in Arabidopsis and tree species (reviewed by Boerjan et al., 2003). Efforts have been made to increase the flavonoid and chlorogenic acid content of tomatoes to create ‘functional food’ with higher antioxidant levels (Muir et al., 2001; Niggeweg et al., 2004). Metabolic engineering for disease resistance has been focused so far on fungal pathogens (Punja, 2001). The most successful and widespread applied approach is the expression of the stilbene synthase gene from grapevine or peanut in heterologous plant species. Increased fungal resistance has been obtained in tobacco, tomato, rice, barley, wheat, alfalfa, papaya, and aspen (reviewed by Punja, 2001; Zhu et al., 2004). The overexpression of the gene coding for isoflavone O-methyltransferase (OMT) in alfalfa increases the levels of the phytoalexin medicarpin and increases resistance against fungal infection (He and Dixon, 2000). Finally, it was shown that PAL-overexpressing tobacco plants have improved resistance against fungal infection (Howles et al., 1996; Way et al., 2002; Shadle et al., 2003).
The role of the phenylpropanoid pathway in plant defence mechanisms includes the synthesis of preformed or inducible physical and chemical barriers against infection and the synthesis of signal molecules involved in local and systemic signalling for defence gene induction (Nicholson and Hammerschmidt, 1992; Dixon et al., 2002). Research on phenylpropanoids active in plant defence against nematode infection is limited, compared with fungal infection, although phenolic compounds were already mentioned by Rohde (1972) and Giebel (1982). Levels of phenylpropanoid compounds are higher in nematode-resistant cultivars—chlorogenic acid in tomato and pepper roots and rice (Hung and Rohde, 1973; Plowright et al., 1996; Pegard et al., 2005), isoflavonoids and medicarpin in alfalfa roots (Edwards et al., 1995; Balbridge et al., 1998)—or increase as a result of nematode infection (Mahmood and Saxena, 1986; Huang and Barker, 1991; Cook et al., 1995; Kennedy et al., 1999). It has also been shown that enzymes of the phenylpropanoid biosynthetic pathway are induced in plants after nematode infection (Edens et al., 1995; Balbridge et al., 1998). Bioassays involving natural plant products, including phenylpropanoids, have shown the existence of potential chemical barriers to nematode infection among these compounds (Chitwood, 2002; Wuyts et al., 2006). Lignin is a potential physical barrier to nematode infection as it is known that the endodermis of the vascular cylinder in plant roots is not crossed by most endoparasitic nematodes, except cyst nematodes, because of lignification.
Engineering for resistance against nematodes in plants has, so far, not involved phenylpropanoid metabolism (Atkinson et al., 2003). As such, potential phytoanticipins or phytoalexins have not been tested in vivo by (over)production of these compounds in plants. Therefore, mutants and transgenics with an altered phenylpropanoid metabolism are interesting candidates for nematode infection and reproduction tests. They include plants with alterations in the lignin biosynthetic pathway (Atanassova et al., 1995; Meyer et al., 1998), in the flavonoid pathway (Goldsbrough et al., 1996; He and Dixon, 2000; Muir et al., 2001; Peer et al., 2001), and in the ‘core’ part of the phenylpropanoid pathway with effects on lignin and flavonoid metabolism (Howles et al., 1996; Blount et al., 2000; Borevitz et al., 2000). As such, the perspectives of metabolic engineering for resistance against plant parasitic nematodes can be explored. Moreover, it could become clear which part of the phenylpropanoid pathway is utilized in defence or which part is required by nematodes for successful infection and establishment in plants.
The specific objective of the study was to evaluate infection and reproduction of plant parasitic nematodes in mutant and transgenic Arabidopsis and tobacco plants in comparison with their wild types. Before the actual infection with nematodes, roots of these plants were histochemically stained or root extracts were analysed by liquid chromatography to confirm the altered profile of metabolites as described for leaf and stem tissue. Infection tests were performed with the root knot nematode Meloidogyne incognita, which is a sedentary endoparasite. Juvenile nematodes infect roots in the elongation zone just behind the root tip and migrate intercellularly through the cortex towards the root apex. Once they have arrived, they turn round towards the vascular cylinder, where they induce cell (re)differentiation processes to establish a permanent feeding site, which functions as a nutrient sink in host roots (Wyss et al., 1992; Gravato Nobre et al., 1995). Nematodes are serious threats to agricultural production worldwide. Current control measures still rely mostly on a few expensive, unsafe, and environmentally hazardous chemical pesticides. Natural or transgenic nematode-resistant cultivars are promising alternatives as part of an integrated pest management strategy for large-scale as well as small-scale cultivation.
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Transgenic and mutant plants
In Table 1 a summarized description is provided of the mutant and transgenic plants that were included in the nematode infection tests. Seeds of the different mutant and transgenic plant lines were obtained as follows: Arabidopsis C4H-F5H and control plants from Dr Clint Chapple (Department of Biochemistry, Purdue University, West Lafayette, IN, USA); tobacco OMT antisense line B10 from Dr Michel Legrand (Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique, Strasbourg, France); tobacco PAP2 and control plants from Dr Justin Borevitz (Plant Biology, Salk Institute, La Jolla, CA, USA); tobacco PAL-sense line 10-6-OX434 plants and control line C17 plants from Dr Richard Dixon (Plant Biology, The Samuel Roberts Noble Foundation, Inc., Ardmore, OK, USA); Arabidopsis tt3 (line 84), tt4 (line UV118a), tt5 (line 86), and tt7 (line 88) and Ler from Dr Wendy Peer (Department of Horticulture, Purdue University) and Dr Maarten Koornneef (Plant Sciences, Wageningen University, Wageningen, The Netherlands). The tt4 (line UV118a) was originally a gift of Dr Brenda Winkel (Fralin Center for Biotechnology, Virginia Tech, Blacksburg, VA, USA) to Dr Wendy Peer.
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Plant growth
Nematode infection tests were performed in vitro. Tobacco and Arabidopsis seeds were surface-sterilized by a 1 min soak in 70% ethanol, followed by 2 min in 0.8% NaOCl with 0.1% Tween-20, and excessive rinsing in water. Seeds were plated on MS-medium (Murashige and Skoog, 1962) (Duchefa Biochemie, Haarlem, The Netherlands) containing 1% sucrose and 0.8% agar for germination. Seven-day-old seedlings of transformed, mutant, and control plants were randomly transferred to modified KNOP medium (Sijmons et al., 1991) in six-well tissue culture plates (Greiner Bio-One, Wemmel, Belgium), with each plate containing one or two plants of each type. Twelve seedlings per plant type were included in the infection tests and at least three independent tests were done. Seedlings were grown at 23 °C with a 16 h light/8 h dark cycle. When plants had grown a sufficient root system (1 week on KNOP medium), they were inoculated with nematodes.
Confirmation of alterations in phenylpropanoid metabolism in roots
Only for the tt-mutants has the phenylpropanoid profile been analysed in roots (Peer et al., 2001). For the other plants, analysis has been performed on leaves or stem tissue only. Also only in-vivo grown (growth chamber/greenhouse) plants have been analysed for phenylpropanoids, while the nematode infection tests were performed in vitro. Therefore, roots of in vitro-grown transformed, mutant, and wild-type plants of tobacco and Arabidopsis were analysed either by HPLC or histochemical staining at the time of nematode inoculation.
HPLC analysis was performed on methanol extracts of fresh tissues of in vitro-grown tobacco. Approximately 500 mg of stems and leaves or 50 mg of roots of PAL-sense and control plants were ground with a small pestle in 1 ml methanol and extracted for 5 min at room temperature under constant shaking. For the PAP2 and control plants 
200 mg of stems and leaves or 30 mg of roots were extracted in 1 ml methanol for 15 min. Samples were passed through 0.45 µm PTE filters (Machery-Nagel, Düren, Germany). HPLC analysis was carried out on a HP1100 system (Hewlett Packard, Waldbronn, Germany) using an Inertsil ODS2 (250x3 mm, particle size 5 µm) RP18 analytical column (Chrompack, Middelburg, The Netherlands). Extracts (15 µl) were separated at 25 °C at a flow rate of 0.4 ml min–1 using a gradient of acetonitrile in water (MilliQ water at pH 3.0 with phosphoric acid): 12% for 10 min, 18% for 5 min, 45% for 20 min, 100% for 7 min, and a step back to 12% for 10 min. A diode array detector (Hewlett Packard) was used to record the online spectra (254, 280, 330, 350, and 366 nm) of compounds eluting from the column. Peak identification and integration were carried out on HP ChemStations software using phenylpropanoid standards (Sigma-Aldrich, Inc., Bornem, Belgium). Peaks were identified by comparing their retention times and UV absorbance spectra with those of standards. Standard calibration curves were determined for quantification.
Lignin was detected in the roots of Arabidopsis C4H-F5H and Col wild-type plants and in tobacco OMT antisense and Samsun NN wild-type plants by histochemical staining. As fresh sections could not be made of the very thin and transparent in vitro roots of Arabidopsis and tobacco, whole roots were stained in solutions to which detergent (Triton X-100) was added. Plants were removed from agar, gently washed, and submerged in the staining solutions. Both Mäule and Wiesner staining procedures were applied according to standard protocols (Chapple et al., 1992; Atanassova et al., 1995). Stained roots were examined by bright-field microscopy at 40- and 100-fold magnification. Digital photographs were taken with a SPOT RT camera and SPOT software version 3.3 (Diagnostic Instruments, Inc., Sterling Heights, MI, USA).
For the detection of phenylpropanoid compounds, Arabidopsis roots were stained with saturated (0.25%, w/v) diphenylboric acid 2-aminoethyl ester (DPBA) (Sigma-Aldrich, Inc., Bornem, Belgium) with 0.02% (v/v) Triton X-100, and observed under an epifluorescence microscope equipped with a DAPI filter (excitation 340–380 nm, suppression LP 430 nm) and a FITC filter (excitation 450–490 nm, suppression LP 520 nm) (Peer et al., 2001). Photo-documentation of roots was achieved with a SPOT RT camera and SPOT software version 3.3.
Nematode inoculum and infection
Meloidogyne incognita was cultured in vitro on Ri T-DNA-transformed tomato roots (hairy roots) (Verdejo et al., 1988). Egg masses were isolated from roots and allowed to hatch in sterile distilled water for 5–7 d to obtain juvenile nematodes. Depending on the availability, M. incognita populations isolated from tomato (Belgium), banana (Morocco), or fig tree (Spain) were used. Per plant, i.e. in one well of a six-well plate, 10 or 20 juvenile nematodes were inoculated on the culture medium in a small drop of water. The plates were sealed with Parafilm and incubated at 23 °C or 26 °C depending on the nematode population (indicated in Table 2).
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Analysis of infection and reproduction
The infection of plants by nematodes was monitored up to 3 weeks after inoculation by light microscopy. Nematode reproduction was evaluated 10 weeks after inoculation. The number of sedentary females, egg masses, and juveniles in roots and medium were counted by direct observations of the six-well plates on a reverse-contrast light microscope. This is possible because of the transparency of the culture medium and plates and the transparency and thin diameter of the in vitro-cultured roots (Sijmons et al., 1991). Afterwards, the egg masses were hand-picked from the roots and transferred per root system to a 10% commercial bleach solution (0.4% NaOCl) to dissolve the gelatinous matrix (Hussey and Barker, 1973). The number of eggs per egg mass and per root system was then determined.
Statistical analysis
Data on nematode reproduction were analysed using ANOVA (Statistica® Release 6, Statsoft, Inc., Tulsa, OK, USA). For multiple comparisons of group means the Tukey test was applied. Data were log(x+1) transformed prior to analysis to reduce the natural variance in nematode reproduction data. When the conditions for ANOVA—normal distribution and homogeneity of variances—were not met, the non-parametric alternative Kruskal–Wallis analysis of variance by ranks was applied (Statistica®). When the Kruskal–Wallis statistic was significant, multiple comparisons between treatments were calculated as described by Siegel and Castellan (1988).
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Results |
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Nematode infection and reproduction in in vitro-grown Arabidopsis and tobacco
Meloidogyne incognita infected and reproduced in both Arabidopsis and tobacco, but reproduction ratios (final number of nematodes and eggs/inoculated number of nematodes) varied much between repetitions. This was mainly due to the source (or pathotype) of the nematode population, with M. incognita isolated from tomato plants giving the lowest reproduction (Table 2). At 1 d after inoculation, juveniles accumulated in tobacco root tips and after 5 d root knots were visible. In Arabidopsis roots, at 5 d after inoculation, swollen bodies of developing females were noted. Male nematodes occurred rarely. Overall, M. incognita significantly infected and reproduced in both Arabidopsis and tobacco under the test conditions.
Alterations which affect lignin metabolism
The fah1-2 mutant of Arabidopsis thaliana is deficient in ferulic acid 5-hydroxylase (F5H). This enzyme catalyses the irreversible hydroxylation of precursors for guaiacyl lignin towards precursors for syringyl lignin biosynthesis. As such, the mutant only accumulates guaiacyl lignin (Chapple et al., 1992). The gene encoding F5H in Arabidopsis (Meyer et al., 1996) has been used in a transcriptional fusion construct with the promoter of C4H, an early enzyme in the phenylpropanoid pathway, for transformation of the fah1-2 mutant. The resulting C4H-F5H plants contain a lignin that is highly enriched in syringyl monomer units: an average of 76 mol% compared with 20 mol% in wild-type Arabidopsis (Meyer et al., 1998; Marita et al., 1999). Moreover, in wild-type plants, guaiacyl and syringyl lignin monomers are restricted to cells of the vascular bundles and to cells of the sclerified parenchyma that flank the vascular bundles, respectively. In the C4H-F5H plants, syringyl units also replace guaiacyl units in the cells of the vascular bundles (Meyer et al., 1998).
Using the Mäule and Wiesner staining procedures, the result of alterations in the lignin pathway in roots of transgenics was checked. The Mäule reaction is specific for free syringyl units, while the Wiesner stain reflects the total lignin content (syringyl and guaiacyl lignin) as it reacts with cinnamaldehyde residues in lignin (Monties, 1989). The overall changes in the syringyl/guaiacyl ratio that have been described for stem and leaf tissues of C4H-F5H plants were confirmed in roots of these plants when grown under the conditions of the infection tests. The vascular cylinder of roots of the C4H-F5H plants stained very bright red (more than Col wild-type roots), indicative of syringyl lignin, in the treatment with the Mäule reagent. Total lignin was detected in roots of all plants (Fig. 1A).
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Infection of lignin-altered Arabidopsis with M. incognita was repeated four times, including three experiments with the tomato population and one with the fig-tree population. Only in one experiment with the tomato population, was significant reproduction obtained. The numbers of egg masses, juveniles, and the resulting reproduction ratio were significantly lower in the C4H-F5H plants than in the wild-type plants at 10 weeks after inoculation (P <0.01, 0.001) (Table 3). In the experiment with the fig-tree population, much higher reproduction ratios were obtained. Reproduction was again significantly lower in the C4H-F5H plants than in the wild type (P <0.05) (Table 3). The number of egg masses did not differ between plants at 10 weeks after inoculation. The difference lay in the number of juveniles (Table 3) and the number of eggs produced per egg mass (Table 4). The percentage of juveniles in the population was used as an indicator of the time required for the transition between life cycles (egg
mobile juvenile). This was again significantly lower in the C4H-F5H plants (P <0.05). Also the number of developing females, i.e. the nematodes that have become sedentary but have not yet produced an egg mass (sedentaryreproductive), was lower in the C4H-F5H plants (P <0.05) (Table 4).
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The selected tobacco plants are altered in another enzyme of the lignin biosynthesis pathway, OMT, which catalyses the conversions of hydroxycinnamic CoA esters to syringyl lignin units (Guo et al., 2001; Zhong et al., 1998). In the transgenic line with antisense expression, OMT activity in stem tissue is reduced to 8% of the wild-type level, together with a decrease in the syringyl monomer content to 10% of the wild-type level and a syringyl/guaiacyl ratio of 0.07 compared with 0.9–1.1 in the wild type (Atanassova et al., 1995).
Stained lignin was detected in the epi- and endodermis of roots of the wild-type Samsun NN plants. In the endodermis, the lignin polymer contained syringyl units. Staining of OMT antisense roots resulted in no syringyl units and little total lignin in the endodermis compared with the wild type.
Infection of lignin-altered tobacco with M. incognita was repeated three times (Table 5). In one out of three experiments, the average reproduction ratio obtained for the OMT antisense plants was significantly higher than for wild-type plants (P <0.001). Although not significantly different, the same trend was observed in all experiments (Table 5). The number of egg masses and the number of eggs per egg mass were equal among the transgenic and wild-type plants (Table 4). The difference in reproduction lay in the number of juveniles, which was significantly higher in OMT antisense plants (P <0.05, 0.001) in two out of three experiments. As the percentage of juveniles in the final nematode population was higher than in the wild-type plants, the life cycle of M. incognita appeared to be altered in roots of OMT-transgenic plants.
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Alterations which affect lignin and/or flavonoid metabolism
During the evaluation of activation-tagged Arabidopsis, a mutant was observed with intense purple pigmentation in vegetative organs, including roots, throughout development (pap1-D). In this plant a massive activation of the phenylpropanoid pathway occurs due to the constitutive expression of a MYB transcription factor. Enhanced activity of key enzymes in the pathway leads to increased accumulation of hydroxycinnamic acids, syringyl and guaiacyl lignin, and flavonoids, including anthocyanidins. The purple pigmentation has been transferred to tobacco plants by transformation with a CaMV 35S promoter::cDNA fusion construct, containing coding sequences of an expressed sequence tag (PAP2) with high homology to the coding sequence of PAP1, which is a MYB transcription factor (Borevitz et al., 2000). No quantitative studies of enzyme activities or phenylpropanoid levels have been performed before on PAP2 tobacco plants.
PAP2 tobacco plants, grown under in vitro conditions, showed the intense purple pigmentation, indicative for anthocyanidins, as has been described for in vivo-grown plants. Roots were purple as well (Fig. 1B). In methanol extracts of root tissue, mainly chlorogenic acid and a glycoside of 4-coumaric acid were detected by HPLC analysis. In stem and leaf tissue, two other phenylpropanoids, caffeic acid and rutin (a glycoside of the flavonol quercetin), were present. Levels of chlorogenic acid were higher in root tissue than in stem and leaf tissues. PAP2 roots contained 3.5-fold more chlorogenic acid than roots of wild-type Xanthi, while 4-coumaric acid was not detected any more. Levels of caffeic acid and rutin were also higher in PAP2 stem and leaf tissue (Table 6).
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Meloidogyne incognita infected PAP2 and Xanthi wild-type tobacco plants under in vitro conditions (Table 7). In three independent experiments, reproduction was significantly higher in the PAP2 plants than in the wild-type control plants (P <0.01, 0.001). The number of egg masses did not differ, but the number of eggs per egg mass and the number of juveniles was significantly higher in the PAP2 plants (P <0.05, 0.001) (Tables 4, 7). The percentage of juveniles in the population was identical in the control and transgenic plants, which means that the life cycle of M. incognita was not affected.
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The PAL-sense plants are first generation (T1) overexpressor plants from a primary transformant (T0) harbouring a heterologous (bean) phenylalanine ammonia-lyase (PAL) gene with CaMV 35S enhancer sequences in its promoter. PAL is the enzyme which catalyses the first step in the phenylpropanoid pathway: the conversion of L-phenylalanine into cinnamic acid. The T0 showed homology-dependent silencing of the endogenous tobacco PAL genes, which resulted in reduced PAL activity and lower levels of phenylpropanoids, including chlorogenic acid and rutin (Elkind et al., 1990). In the T1 plants PAL-activity is up to 5- and 2-fold greater in leaf and stem tissue, respectively, compared with wild-type levels, due to the transgene-encoded PAL. In leaf tissue, this results in increased levels of chlorogenic acid and a 4-coumaric acid glycoside. Levels of rutin are not increased which suggests that additional flux control points exist in the flavonoid pathway (Howles et al., 1996). PAL overexpression has little effect on the total lignin content or on lignin composition in stem tissue (Sewalt et al., 1997).
PAL sense tobacco plants showed higher levels of all four phenylpropanoids in stem and leaf tissue compared with control plants. In root tissue the level of chlorogenic acid was twice as high. The 4-coumaric acid glycoside was still detected and levels were similar to control roots (Table 6). Control plants for the PAL sense plants were the first-generation progeny line that had lost the bean PAL transgene through segregation (Sewalt et al., 1997).
Unlike for the PAP2 plants, reproduction in the PAL sense plants was not different from the control in three independent experiments (data not shown).
Alterations which affect flavonoid metabolism
The flavonoid accumulation patterns of Arabidopsis transparent testa (tt) mutants are well described and result from defects in different intermediate steps in the flavonoid biosynthetic pathway (Koornneef, 1981, 1990; Koornneef et al., 1982; Shirley et al., 1995). A deficiency in a pathway enzyme results in the accumulation of precursors.
The flavonoid profiles in the roots of tt-mutants and Ler plants that have been described by Peer et al. (2001) were confirmed for plants grown under in vitro conditions. In 2–7-d-old seedlings flavonoids accumulate at specific sites along the root axis, i.e. at sites of organ transition and at the root tip (Peer et al., 2001). In the roots of the plants in the present study, which were 14-d-old, flavonoids were also detected more randomly along the root axis (Fig. 1C).
Infection tests with M. incognita (fig tree) were repeated in four independent experiments. Nematodes reproduced well in all plants (average reproduction ratio of 59), but no significant differences (P <0.05) in reproduction between tt-mutants or between mutants and Ler occurred (data not shown).
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Discussion |
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Based on nematode feeding behaviour, it is possible that infection and reproduction by root knot nematodes is affected by changes in lignin content or composition. These nematodes establish their permanent feeding sites inside the vascular cylinder, which is the main site of secondary wall formation and lignification in roots. In C4H-F5H Arabidopsis plants, infection by M. incognita was not affected by the transgenic phenylpropanoid profile as the number of egg masses was equal to wild-type Col plants. Fecundity and development, however, were significantly reduced compared with the wild type: fewer eggs were produced per egg mass, fewer eggs hatched, and fewer juveniles developed into sedentary females. Meloidogyne incognita juveniles select procambial cells in the differentiation zone at the entry of the vascular cylinder, just above the root tip, to initiate the development of giant cells (permanent feeding site). As the root matures, cells of the vascular cylinder undergo secondary wall thickening. At this point, juveniles are already feeding from the initiated giant cells (Gravato Nobre et al., 1995). In wild-type Arabidopsis, the sclerified parenchyma of the vascular cylinder contains mainly syringyl lignin, while the xylem bundles only contain guaiacyl lignin. In the vascular cylinder of C4H-F5H plants, lignified tissue contains significantly more syringyl units, and this syringyl lignin also replaces guaiacyl lignin in the xylem bundles (Meyer et al., 1998). The increased levels of syringyl lignin in the vascular bundles possibly impede the flow of nutrients to the nematode's giant cells or hamper the nematode in feeding from its giant cells, which leads to reduced reproduction, a reduced ‘quality’ of eggs and overall a change in the development rate. Bell (1981) stated that the welfare of sedentary nematodes depends on the establishment and maintenance of the giant cells. In naturally resistant cultivars, nematode numbers often do not decline rapidly after infection. Instead, the nematode's development rate is slowed as fewer females develop egg masses and each mass contains fewer eggs. The transgenic C4H-F5H Arabidopsis plants seemed to affect M. incognita reproduction accordingly. In in vitro bioassays, sinapic acid and syringic acid inhibit motility of M. incognita juveniles, but the effect is limited and only temporary. Neither of these compounds affects hatching (Wuyts et al., 2006).
Arabidopsis and tobacco have been altered with enzymes of the lignin biosynthetic pathway to increase knowledge on control points for lignin content and lignin composition with respect to improved paper pulping and forage digestibility. Guaiacyl units in the lignin polymer are monomethoxylated, which means that the C-5 aromatic position is available for carbon–carbon linkage with, for example, another guaiacyl unit during the polymerization process. As syringyl units are dimethoxylated, syringyl-containing lignin has fewer 5-5' linkages and is therefore less condensed. For the paper industry, this means that wood containing this type of lignin (angiosperm ‘hard’ wood) is easier to delignify (Boudet et al., 1995). On the other hand, syringyl-rich lignins are more linear and penetrate a larger fraction of the cell wall. This limits the digestibility of polysaccharides by hydrolytic enzymes and decreases the nutritional value of forages (Jung and Deetz, 1993). In relation to plant pathogens, it has been shown that induced lignification in response to fungal infection is accompanied by an increased syringyl content in wheat leaves (Ride, 1975) and increased levels of sinapyl alcohol dehydrogenase activity in banana roots (de Ascensao and Dubery, 2000). Cellulases and pectinases have been detected in secretions of nematodes, including Meloidogyne spp. (Williamson and Gleason, 2003), but a relationship between lignin composition in cell walls and susceptibility to degradation by nematode enzymes has not been reported. Also no correlation has been established between the level of lignification in the vascular cylinder and the metabolic sink capacity of nematode giant cells nor the capacity of nematodes to feed on their giant cells in mature roots.
In roots of OMT antisense tobacco plants with a reduced syringyl lignin content, the life cycle of M. incognita progressed faster than in wild-type plants, as significantly more eggs had developed into juvenile nematodes. This contrasts nicely with the effect of increased levels of syringyl lignin in Arabidopsis plants. The number of egg masses was not higher in OMT-suppressed plants, probably because the decrease in syringyl lignin did not affect giant cell induction or because roots of in vitro plants simply do not sustain higher infection levels.
Roots of both PAP2 and PAL sense plants contained higher levels of chlorogenic acid than those of control plants. The 4-coumaric acid glycoside was not detected in roots of PAP2 plants, while PAL sense roots contained similar levels as control roots. Only in PAP2 roots was reproduction of M. incognita stimulated, which means that the 4-coumaric acid glycoside negatively affected reproduction or that metabolites other than chlorogenic acid in PAP2 affected reproduction in a positive sense. The purple colour of the PAP2 roots is an indication of the accumulation of other compounds, including anthocyanidins, as in stem tissue (Borevitz et al., 2000). The analysis of anthocyanidins and related compounds would have required other extraction and detection methods. In PAL sense plants the effects of increased PAL activity are believed to be limited to increased levels of chlorogenic acid and 4-coumaric acid in stem tissue (Howles et al., 1996).
Infection tests with Arabidopsis tt-mutants have the potential of providing information on the involvement of flavonoids in infection and reproduction of nematodes. Flavonoids affect nematode behaviour in in vitro bioassays, including chemotaxis and motility (Wuyts et al., 2006). Indirect effects may arise from auxin transport inhibition in plants by flavonoids which results in local auxin accumulation. Flavonoids act on auxin accumulation by inhibiting or stimulating auxin turnover (Mathesius, 2001) or by modulating auxin transport (Peer et al., 2004). Feeding-site induction by sedentary (cyst and root knot) nematodes requires cell cycle activation, which itself is regulated by the phytohormones auxin and cytokinin (Goverse et al., 2000). Flavonoids are candidate modulators of local auxin manipulation in nematode infection mechanisms.
No significant differences in infection and reproduction of M. incognita were observed between tt mutants and wild-type plants or between the different tt mutants. However, between the six-well plates of each experiment, results varied considerably. Other test methods, including the growth and infection of individual plants in soil, may still be considered to test effects of altered flavonoid profiles.
Metabolically engineered and mutant Arabidopsis and tobacco with altered phenylpropanoid pathways were infected with the sedentary root knot nematode M. incognita to assess the effect of the transgene or mutation on the infection and reproduction of nematodes. Besides the ones that were used in this study, a large number of other plants have been designed with different or complementing alterations in the phenylpropanoid pathway of secondary metabolism, which can be tested and contribute to the knowledge on plant–nematode interactions.
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Acknowledgements |
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Work reported in this paper was supported by the ‘Institute for the Promotion of Innovation through Science and Technology in Flanders’ (IWT-Vlaanderen), Belgium. Providers of seeds of mutant and transgenic lines are gratefully acknowledged. Comments on the manuscript by Bruno Cammue were much appreciated.
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Abbreviations |
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C4H, cinnamic acid 4-hydroxylase; F5H, ferulic acid 5-hydroxylase; OMT, O-methyltransferase; PAL, L-phenylalanine ammonia-lyase; tt, transparent testa.
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Atanassova R, Favet N, Martz F, Chabbert B, Tollier MT, Monties B, Fritig B, Legrand M. (1995) Altered lignin composition in transgenic tobacco expressing O-methyltransferase sequences in sense and antisense orientation. The Plant Journal 8:465–477.[CrossRef][Web of Science]
Atkinson HJ, Urwin PE, McPherson MJ. (2003) Engineering plants for nematode resistance. Annual Review of Phytopathology 41:615–639.[CrossRef][Web of Science][Medline]
Balbridge GD, O'Neill NR, Samac DA. (1998) Alfalfa (Medicago sativa L.) resistance to root-lesion nematode, Pratylenchus penetrans: defense-response gene mRNA and isoflavonoid phytoalexin levels in roots. Plant Molecular Biology 38:999–1010.[CrossRef][Web of Science][Medline]
Bell AA. (1981) Biochemical mechanisms of disease resistance. Annual Review of Plant Physiology 32:21–81.[Web of Science]
Blount JW, Korth KL, Masoud SA, Rasmussen S, Lamb C, Dixon RA. (2000) Altering expression of cinnamic acid 4-hydroxylase in transgenic plants provides evidence for a feedback loop at the entry point into the phenylpropanoid pathway. Plant Physiology 122:107–116.
Boerjan W, Ralph J, Baucher M. (2003) Lignin biosynthesis. Annual Review of Plant Biology 54:519–546.[CrossRef][Medline]
Borevitz JO, Xia Y, Blount J, Dixon RA, Lamb CJ. (2000) Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis. The Plant Cell 12:2383–2393.
Boudet AM, Lapierre C, Grima-Pettenati J. (1995) Biochemistry and molecular biology of lignification. New Phytologist 129:203–236.[CrossRef]
Chapple CCS, Vogt T, Ellis BE, Somerville CR. (1992) An Arabidopsis mutant defective in the general phenylpropanoid pathway. The Plant Cell 4:1413–1424.
Chitwood DJ. (2002) Phytochemical based strategies for nematode control. Annual Review of Phytopathology 40:221–249.[CrossRef][Web of Science][Medline]
Cook R, Tiller SA, Mizen KA, Edwards R. (1995) Isoflavonoid metabolism in resistant and susceptible cultivars of white clover infected with the stem nematode Ditylenchus dipsaci. Journal of Plant Physiology 146:348–354.
de Ascensao ARDCF and Dubery IA. (2000) Panama disease: cell wall reinforcement in banana roots in response to elicitors from Fusarium oxysporum f.sp. cubense race four. Phytopathology 90:1173–1180.[Medline]
Dixon RA, Achnine L, Kota P, Liu CJ, Reddy MSS, Wang L. (2002) The phenylpropanoid pathway and plant defence: a genomics perspective. Molecular Plant Pathology 3:371–390.[CrossRef]
Edens RM, Anand SC, Bolla RI. (1995) Enzymes of the phenylpropanoid pathway in soybean infected with Meloidogyne incognita or Heterodera glycines. Journal of Nematology 27:292–303.
Edwards R, Mizen T, Cook R. (1995) Isoflavonoid conjugate accumulation in the roots of lucerne (Medicago sativa) seedlings following infection by the stem nematode (Ditylenchus dipsaci). Nematologica 41:51–66.
Elkind Y, Edwards R, Mavandad M, Hedrick SA, Ribak O, Dixon RA, Lamb CJ. (1990) Abnormal plant development and down-regulation of phenylpropanoid biosynthesis in transgenic tobacco containing a heterologous phenylalanine ammonia-lyase gene. Proceedings of the National Academy of Sciences, USA 87:9057–9061.
Giebel J. (1982) Mechanisms of resistance to plant nematodes. Annual Review of Phytopathology 20:257–279.[CrossRef][Web of Science]
Goldsbrough AP, Tong Y, Yoder JI. (1996) Lc as a non-destructive visual reporter and transposition excision marker gene for tomato. The Plant Journal 9:927–933.[CrossRef]
Goverse A, de Almeida Engler J, Verhees J, van der Krol S, Helder J, Gheysen G. (2000) Cell cycle activation by plant parasitic nematodes. Plant Molecular Biology 43:747–761.[CrossRef][Web of Science][Medline]
Gravato Nobre MJ, von Mende N, Dolan L, Schmidt KP, Evans K, Mulligan B. (1995) Immunolabelling of cell surfaces of Arabidopsis thaliana roots following infection by Meloidogyne incognita (Nematoda). Journal of Experimental Botany 46:1711–1720.
Guo D, Chen F, Inoue K, Blount JW, Dixon RA. (2001) Down-regulation of caffeic acid 3-O-methyltransferase and caffeoyl CoA 3-O-methyltransferase in transgenic alfalfa: impacts on lignin structure and implications for the biosynthesis of G and S lignin. The Plant Cell 13:73–88.
He XZ and Dixon RA. (2000) Genetic manipulation of isoflavone 7-O-methyltransferase enhances biosynthesis of 4'-O-methylated isoflavonoid phytoalexins and disease resistance in alfalfa. The Plant Cell 12:1689–1702.
Howles PA, Sewalt VJH, Paiva NL, Elkind Y, Bate NJ, Lamb CJ, Dixon RA. (1996) Overexpression of L-phenylalanine ammonia-lyase in transgenic tobacco plants reveals control points for flux into phenylpropanoid biosynthesis. Plant Physiology 112:1617–1624.[Abstract]
Huang JS and Barker KR. (1991) Glyceollin l in soybean–cyst nematode interactions: spatial and temporal distribution in roots of resistant and susceptible soybeans. Plant Physiology 96:1302–1307.
Hung CL and Rohde RA. (1973) Phenol accumulation related to resistance in tomato to infection by root knot and lesion nematodes. Journal of Nematology 5:253–258.
Hussey RS and Barker KRA. (1973) A comparison of methods of collecting inocula of Meloidogyne spp, including a new technique. Plant Disease Reporter 57:1025–1028.[Web of Science]
Jung HG and Deetz DA. (1993) Cell wall lignification and degradability. In Jung HG, Buxton DR, Hatfield RD, Ralph J (Eds.). Forage cell wall structure and digestibility (ASA-CSSA-SSSA, Madison, WI) pp. 315–346.
Jung W, Yu O, Lau SMC, O'Keefe DP, Odell J, Fader G, McGonigle B. (2000) Identification and expression of isoflavone synthase, the key enzyme for biosynthesis of isoflavones in legumes. Nature Biotechnology 18:208–212.[CrossRef][Web of Science][Medline]
Kennedy MJ, Niblack TL, Krishnan HB. (1999) Infection by Heterodera glycines elevates isoflavonoid production and influences soybean nodulation. Journal of Nematology 31:341–347.
Koornneef M. (1981) The complex syndrome of ttg mutants. Arabidopsis Information Service 18:45–51.
Koornneef M. (1990) Mutations affecting the testa color in Arabidopsis. Arabidopsis Information Service 27:1–4.
Koornneef M, Luiten W, de Vlaming P, Schram AW. (1982) A gene controling flavonoid-3'-hydroxylation in Arabidopsis. Arabidopsis Information Service 19:113–115.
Mahmood I and Saxena SK. (1986) Relative susceptibility of different cultivars of tomato to Rotylenchulus reniformis in relation to changes in phenolics. Revue de Nématologie 9:89–91.[Medline]
Marita JM, Ralph J, Hatfield RD, Chapple C. (1999) NMR characterization of lignins in Arabidopsis altered in the activity of ferulate 5-hydroxylase. Proceedings of the National Academy of Sciences, USA 96:12328–12332.
Mathesius U. (2001) Flavonoids induced in cells undergoing nodule organogenesis in white clover are regulators of auxin breakdown by peroxidase. Journal of Experimental Botany 52:419–426.
Meyer K, Cusumano JC, Somerville CR, Chapple CCS. (1996) Ferulate-5-hydroxylase from Arabidopsis thaliana defines a new family of cytochrome P450-dependent monooxygenases. Proceedings of the National Academy of Sciences, USA 93:6869–6874.
Meyer K, Shirley AM, Cusumano JC, Bell-Lelong DA, Chapple CCS. (1998) Lignin monomer composition is determined by the expression of a cytochrome P450-dependent monooxygenase in Arabidopsis. Proceedings of the National Academy of Sciences, USA 95:6619–6623.
Monties B. (1989) Lignins. In Dey PP and Harborne JB (Eds.). Methods in plant biochemistry (Academic Press, London) Vol. 1:. pp. 113–157.
Muir SR, Collins GJ, Robinson S, Hughes S, Bovy A, De Vos CHR, van Tunen AJ, Verhoeyen ME. (2001) Overexpression of petunia chalcone isomerase in tomato results in fruit containing increased levels of flavonols. Nature Biotechnology 19:470–474.[CrossRef][Web of Science][Medline]
Murashige T and Skoog F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiologia Plantarum 15:473–497.[CrossRef]
Nicholson RL and Hammerschmidt R. (1992) Phenolic compounds and their role in disease resistance. Annual Review of Phytopathology 30:369–389.[Web of Science]
Niggeweg R, Michael AJ, Martin C. (2004) Engineering plants with increased levels of the antioxidant chlorogenic acid. Nature Biotechnology 22:746–754.[CrossRef][Web of Science][Medline]
Peer WA, Bandyopadhyay A, Blakeslee JJ, Makam SN, Chen RJ, Masson PH, Murphy AS. (2004) Variation in expression and protein localization of the PIN family of auxin efflux facilitator proteins in flavonoid mutants with altered auxin transport in Arabidopsis thaliana. The Plant Cell 16:1898–1911.
Peer WA, Brown DE, Tague BW, Muday GK, Taiz L, Murphy AS. (2001) Flavonoid accumulation patterns of transparent testa mutants of Arabidopsis. Plant Physiology 126:536–548.
Pegard A, Brizzard G, Fazari A, Soucaze O, Abad P, Dijan-Caporalino C. (2005) Histological characterization of resistance to different root-knot nematode species related to phenolics accumulation in Capsicum annuum. Phytopathology 95:158–165.[Medline]
Plowright RA, Grayer RJ, Gill JR, Rahman ML, Harborne JB. (1996) The induction of phenolic compounds in rice after infection with the stem nematode Ditylenchus angustus. Nematologica 42:564–578.
Punja ZK. (2001) Genetic engineering of plants to enhance resistance to fungal pathogens: a review of progress and future prospects. Canadian Journal of Plant Pathology 23:216–235.
Ride JP. (1975) Lignification in wounded wheat leaves in response to fungi and its possible role in resistance. Physiological Plant Pathology 5:125–134.
Rohde RA. (1972) Expression of resistance in plants to nematodes. Annual Review of Phytopathology 10:233–252.[CrossRef][Web of Science]
Sewalt VJH, Ni W, Blount JW, Jung HG, Masoud SA, Howles PA, Lamb C, Dixon RA. (1997) Reduced lignin content and altered lignin composition in transgenic tobacco down-regulated in expression of L-phenylalanine ammonia-lyase or cinnamate 4-hydroxylase. Plant Physiology 115:41–50.[Abstract]
Shadle GL, Wesley SV, Korth KL, Chen F, Lamb C, Dixon RA. (2003) Phenylpropanoid compounds and disease resistance in transgenic tobacco with altered expression of L-phenylalanine ammonia-lyase. Phytochemistry 64:153–161.[CrossRef][Web of Science][Medline]
Shirley BW, Kubasek WL, Storz G, Bruggemann E, Koornneef M, Ausubel FM, Goodman HM. (1995) Analysis of Arabidopsis mutants deficient in flavonoid biosynthesis. The Plant Journal 8:659–671.[CrossRef][Web of Science][Medline]
Siegel S and Castellan NJ. (1988) Nonparametric statistics for the behavioral sciences (McGraw-Hill Book Co, New York, NY).
Sijmons PC, Grundler FMW, von Mende N, Burrows PR, Wyss U. (1991) Arabidopsis thaliana as a new model host for plant parasitic nematodes. The Plant Journal 1:245–254.[CrossRef][Web of Science]
Verdejo S, Jaffee BA, Mankau R. (1988) Reproduction of Meloidogyne javanica on plant roots genetically transformed by Agrobacterium rhizogenes. Journal of Nematology 20:599–604.
Way HM, Kazan K, Mitter N, Goulter KC, Birch RG, Manners JM. (2002) Constitutive expression of a phenylalanine ammonia-lyase gene from Stylosanthes humilis in transgenic tobacco leads to enhanced disease resistance but impaired plant growth. Physiological and Molecular Plant Pathology 60:275–282.
Williamson VM and Gleason CA. (2003) Plant–nematode interactions. Current Opinion in Plant Biology 6:327–333.[CrossRef][Web of Science][Medline]
Wuyts N, Swennen R, De Waele D. (2006) Effects of plant phenylpropanoid pathway products and selected terpenoids and alkaloids on the behaviour of the plant-parasitic nematodes Radopholus similis, Pratylenchus penetrans and Meloidogyne incognita. Nematology 8:89–101.[CrossRef]
Wyss U, Grundler FMW, Münch A. (1992) The parasitic behaviour of second-stage juveniles of Meloidogyne incognita in roots of Arabidopsis thaliana. Nematologica 38:98–111.[Web of Science]
Yu O, Jung W, Shi J, Croes RA, Fader GM, McGonigle B, Odell JT. (2000) Production of the isoflavones genistein and daidzein in non-legume dicot and monocot tissues. Plant Physiology 124:781–793.
Zhong R, Morrison WH, Negrel J, Ye ZH. (1998) Dual methylation pathways in lignin biosynthesis. The Plant Cell 10:2033–2045.
Zhu YJ, Agbayami R, Jackson MC, Tang CS, Moore PH. (2004) Expression of the grapevine stilbene synthase gene VST1 in papaya provides increased resistance against diseases caused by Phytophthora palmivora. Planta 220:241–250.[CrossRef][Web of Science][Medline]
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