JXB Advance Access originally published online on October 18, 2006
Journal of Experimental Botany 2006 57(15):4015-4023; doi:10.1093/jxb/erl172
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RESEARCH PAPER |
Arbuscular mycorrhiza induces gene expression of the apoplastic invertase LIN6 in tomato (Lycopersicon esculentum) roots
1Leibniz-Institut für Pflanzenbiochemie (IPB), Weinberg 3, D-06120 Halle (Saale), Germany
2Julius-von-Sachs-Institut für Biowissenschaften, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany
* To whom correspondence should be addressed. E-mail: bhause{at}ipb-halle.de
Received 16 June 2006; Accepted 29 August 2006
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Abstract |
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Extracellular invertases are suggested to play a crucial role in the arbuscular mycorrhiza (AM) symbiosis to fulfil the increased sink function of the mycorrhizal root and the supply of the obligate biotrophic AM fungus with hexoses. In tomato (Lycopersicon esculentum), LIN6 represents an apoplastic invertase which is described as a key enzyme in establishing and maintaining sink metabolism. In this study, transcript levels of LIN6 were analysed in tomato roots colonized with the AM fungus Glomus intraradices. Using real-time RT–PCR, a nearly 3-fold increase in LIN6 mRNA levels was detected at late stages of mycorrhization (11 weeks after inoculation). A 1.8-fold induction could already be achieved at earlier stages (5 weeks after inoculation) using higher inoculum concentrations, whereas wounding of non-mycorrhizal roots resulted in up to 12-fold enhanced LIN6 transcripts. As revealed by in situ hybridization, the expression of LIN6 upon mycorrhization was specifically restricted to colonized cells and to the central cylinder. Such a strongly localized pattern due to mycorrhizal cells and to the central core could also be shown for promoter activity using transgenic Nicotiana tabacum plants expressing the gene coding for ß-glucuronidase under the control of the LIN6 promoter. The moderate induction of LIN6 expression in mycorrhizal tomato roots compared with stress-stimulated induction suggested a fine-tuning in the activation of sink metabolism in the mutualistic interaction, avoiding stress-induced defence reactions.
Key words: Apoplastic invertase LIN6, arbuscular mycorrhiza, in situ hybridization, Lycopersicon, real-time RT-PCR, transgenic Nicotiana
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Introduction |
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Arbuscular mycorrhiza (AM) represents an important and widespread symbiosis, formed between most terrestrial plants and zygomycete fungi of the phylum Glomeromycota. This mutualistic association is characterized by an exchange of nutrients. The fungus assists the plant with the acquisition of mineral nutrients, mainly phosphate, from the soil, whereas the plant supplies the fungus with carbon (Harrison, 1998). Previous studies using isotopic labelled substrates revealed the ability of the fungus to take up and metabolize hexoses, preferentially glucose, within intraradical structures (Shachar-Hill et al., 1995; Solaiman and Saito, 1997; Pfeffer et al., 1999). An uptake of hexoses by extraradical hyphae could not be detected (Pfeffer et al., 1999). Nevertheless, it is still a matter of debate whether arbuscules or intercellular hyphae are more important for carbon uptake (for reviews see Bago, 2000; Douds et al., 2000). Whereas the ability of isolated intraradical hyphae to take up glucose has been shown (Solaiman and Saito, 1997), other studies revealed a dependency of the incorporation of labelled glucose into trehalose and glycogen on the presence of arbuscules (Pfeffer and Shachar-Hill, 1996). Moreover, a correlation between arbuscule and spore formation was found (Douds, 1994). The relevance of arbuscules for the uptake of carbohydrates is further supported by the observation that arbuscules are established mainly in the innermost cortex cells near to the central core, suggesting an optimal position for the exchange of metabolites including carbohydrates (Blee and Anderson, 1998).
To supply mycorrhizal roots with hexoses, photosynthetically fixed carbon, which is translocated to the plant sink organs in the form of sucrose, has to be cleaved either by a cytosolic sucrose synthase or by invertases. Plant invertases can be divided into three types characterized by their optimal pH value and subcellular location: (i) the soluble alkaline invertases, which have been associated with the cytoplasm; (ii) the soluble acid invertases residing in the vacuole; and (iii) the apoplastic acid invertases, ionically bound to the cell wall (for reviews see Tymowska-Lalanne and Kreis, 1998; Sturm, 1999; Roitsch and González, 2004). Until now, only a few studies have revealed increased transcript accumulation or activities for sucrose-cleaving enzymes upon mycorrhization. Among the findings were enhanced transcript levels of a cytosolic invertase and sucrose synthase in arbusculated cells of Phaseolus vulgaris (Blee and Anderson, 2002), and transcripts of sucrose synthase encoding genes in mycorrhizal maize roots (Ravnskov et al., 2003) and near mycorrhizal structures in Medicago truncatula (Hohnjec et al., 2003). Regarding enzymatic activity, increases were described for cytosolic invertase in mycorrhizal soybean (Glycine max) (Schubert et al., 2003), and for all types of sucrose-cleaving enzymes, depending on the mycorrhizal stage, in mycorrhizal clover (Trifolium repens) roots (Wright et al., 1998). There, vacuolar invertase and sucrose synthase were induced particularly in the early stages of mycorrhization, cytosolic invertases remained constant, and cell wall-bound apoplastic invertase activity showed a time-dependent increase. Extracellular invertases are suggested to be likely candidates for providing apoplastic hexoses and have a key function in supporting increasing sink strength by phloem unloading (Tymowska-Lalanne and Kreis, 1998; Roitsch et al., 2003). They also seemed to be involved in the carbon allocation in other symbiotic plant–fungi interactions, such as ectomycorrhiza (Salzer and Hager, 1991; Wright et al., 2000). Furthermore, increased activity of acid invertases was also found in interactions with pathogenic biotrophic fungi such as rust fungi (Long et al., 1975; Krishnan and Pueppke, 1988; Tetlow and Farrar, 1992) or powdery mildew fungi (Storr and Hall, 1992; Scholes et al., 1994; Fotopoulos et al., 2003).
In the present study, an analysis was carried out to determine whether apoplastic invertases are involved in the carbon allocation during mycorrhization of tomato plants. In tomato, extracellular invertase isoenzymes are encoded by a gene family including four members: LIN5, LIN6, LIN7, and LIN8. Whereas LIN5 and LIN7 seemed to be mainly involved in the carbohydrate supply of flowers and fruits, respectively (Godt and Roitsch, 1997; Fridman and Zamir, 2003), LIN8 is weakly expressed in roots and leaves (Fridman and Zamir, 2003) as well as in developing fruits (Miron et al., 2002). LIN6 is specifically expressed in actively growing sink tissues such as seedling roots, flower buds, and tumours, and was shown to be induced by glucose and cytokinin as well as stress-related stimuli such as elicitor treatment or wounding, while sucrose synthase was not induced by most of those stimuli (Godt and Roitsch, 1997). Thus, the apoplastic tomato invertase LIN6 was suggested to have an important function in the carbohydrate supply of sink organs as well as in establishing sink metabolism in response to certain external and internal stimuli. Therefore, the transcript accumulation of LIN6 was analysed as the most likely candidate in mycorrhizal roots on a tissue- and cell-specific level.
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Materials and methods |
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Plant material
Wild-type Lycopersicon esculentum cv. Moneymaker and cv. Microtom were obtained from N.L. Chrestensen Erfurter Samen- und Pflanzenzucht (Erfurt, Germany) and Florensis Deutschland GmbH (Weilimdorf/Stuttgart, Germany), respectively. Seeds were germinated in wet expanded clay of 2–5 mm particle size (Original Lamstedt Ton, Fibo ExClay, Lamstedt, Germany) and further cultivated in 8 cm pots filled with the same substrate in a growth chamber at 23 °C, 50% relative humidity, and 16 h light (250 µmol photons m–2 s–1)/8 h dark. Plants were watered with distilled water and fertilized twice per week with 10 ml of Long Ashton (20% phosphate) (Hewitt, 1966). Unless indicated otherwise, experiments were carried out with L. esculentum cv. Moneymaker.
Transgenic Nicotiana tabacum LIN6::uidA plants were selected by sowing on solid MS medium (Duchefa, Haarlem, The Netherlands) containing 50 mg l–1 kanamycin A (Duchefa). After 2 weeks, plants were transferred into pots filled with expanded clay and cultivated as described above for wild-type tomato plants.
Plasmid construction and plant transformation
To generate a translational fusion between the gene encoding ß-glucuronidase (GUS; uidA) and the LIN6 promoter, a 3.4 kb LIN6 promoter fragment was subcloned as a HindIII and XhoI fragment from pR6-11 (Balibrea et al., 2004) into the binary vector pBI101+ (Goetz et al., 2000) to generate pPR6.31, thus fusing the LIN6 start codon in-frame to the uidA translational start site (R Proels, T Roitsch, unpublished data). The LIN6::uidA construct was transformed in tobacco (N. tabacum cv. Samsun NN) using Agrobacterium tumefaciens strain LBA4404 and standard transformation procedures (Horsch et al., 1985). Transgenic lines expressing the LIN6::uidA fusion were characterized by PCR and fluorometric GUS assays (R Proels, T Roitsch, unpublished data).
Inoculation with Glomus intraradices and determination of degree of mycorrhization
The AM fungus Glomus intraradices Schenk & Smith isolate 49 (Maier et al., 1995) was used after enrichment by previous cultivation with leek (Allium porrum cv. Elefant) in expanded clay in the greenhouse. Leeks were grown in clay for at least 4 months and then removed, and the clay was used as an inoculum. Finally, plants were inoculated 4.5 weeks after sowing, except for plants used for invertase in situ staining. Here, 3-week-old plants were inoculated. For inoculation, plants were transferred after careful removal of the previous substrate to new pots of 10–17 cm diameter filled with expanded clay containing at least 10% (v/v) G. intraradices inoculum freshly harvested from mycorrhizal leek plants. Non-mycorrhizal plants were transferred in the same way to pure expanded clay. Transgenic tobacco plants were inoculated using the split root system: roots were divided into two pots; one was filled with pure expanded clay, whereas the other contained 10% (v/v) inoculum. Further cultivation took place in a growth chamber under the same conditions as described above with 25 ml of Long Ashton (20% phosphate) twice per week as fertilization.
Non-mycorrhizal and mycorrhizal roots were harvested at the end of the light period. For the estimation of G. intraradices colonization, a representative cross-section of each root system was taken. Mycorrhizal structures were stained according to Vierheilig et al. (1998) using 5% ink (Sheaffer Skrip jet black, Sheaffer Manufacturing, Madison, WI, USA) in 2% acetic acid. Colonization of root pieces was analysed using a stereomicroscope.
Wounding of roots
For the wounding experiment, roots and leaves of non-mycorrhizal plants of the inoculum dilution series were used 5 weeks after inoculation. Wounding was carried out by squeezing with tweezers. Afterwards, plant material was incubated for up to 10 h in wet tissue paper in the case of roots or by floating on water in the case of leaves.
Determination of invertase activities
Determination of apoplastic, vacuolar, and cytosolic invertase activities was performed according to Wright et al. (1998). For protein extraction, frozen plant material (160±5 mg) was homogenized in liquid nitrogen and incubated with 1.2 ml of extraction buffer for 10 min on ice. After centrifugation (14 000 g, 10 min, 4 °C), the supernatant was used for the determination of vacuolar and cytosolic invertase activities; the pellet was washed three times with extraction buffer and used for activity assay of apoplastic (cell wall-bound) invertases after final resuspension in 1.2 ml of extraction buffer.
The reaction mixture contained 80 µl of extract or resuspended pellet, 80 mM buffer, and 100 mM sucrose in a final volume of 800 µl. As buffer, citrate (pH 4.5) was used for detection of acid apoplastic invertase activity, citrate (pH 5.5) for acid vacuolar invertases, and sodium phosphate (pH 7.5) for alkaline cytosolic invertases. The reaction mixtures were incubated at 37 °C for 0, 30, and 60 min. To stop the reaction, 200 µl of the mixture was taken out, neutralized with 40 µl of TRIS–HCl (pH 10.0), and boiled for 10 min. As controls, protein fractions inactivated by boiling and extraction buffer alone were used. Incubations were carried out in three independent replicates for each sample.
Glucose production of each stopped reaction mixture was measured by an enzyme-linked assay. For this, 30 µl of the stopped reaction mixture was incubated in a microtitre plate with 200 µl of 50 mM imidazole (pH 6.9), 5 mM MgCl2, 2 mM NAD, 1 mM ATP, 0.5 U of glucose-6-phosphatase dehydrogenase from Leuconostoc mesenteroides (Serva, Heidelberg, Germany), and 1 U of hexokinase (Fluka, Buchs, Switzerland). Glucose was used as standard. When the reaction reached a plateau, the absorbance at 340 nm was measured in a 96-well microtitre plate reader (Sunrise, Tecan, Crailsheim, Germany).
Protein content was determined as described by Bradford (1976) using bovine serum albumin (BSA; Carl Roth, Karlsruhe, Germany) as standard.
Real-time RT–PCR analysis
Total RNA of tomato root material was isolated using the Qiagen RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) including a DNase digestion (RNase-free DNase Set, Qiagen). First-strand cDNA synthesis of 1 µg of RNA in a final volume of 20 µl was performed with M-MLV reverse transcriptase, RNase H Minus, Point Mutant (Promega, Madison, WI, USA) according to the supplier's protocol using oligo(dT) (T19) primer. Control reactions were performed by omitting reverse transcriptase.
For real-time PCR, 3 µl of 1:10 diluted cDNA (15 ng of reverse transcribed total RNA) or 3 µl of diluted control reaction were mixed with SYBR Green PCR Mastermix (Applied Biosystems, Warrington, UK), 1 pmol of forward primer, and 1 pmol of reverse primer in a final volume of 10 µl in three independent replicates. For analysis of LIN6 transcript levels (GenBank accession no. AF506005
[GenBank]
), the following primers and annealing temperature were used: forward primer, 5'-ATC TAC CCG TCT AAA G-3'; reverse primer: 5'-CCA ACC AAT ACT CTC C-3'; 48 °C. To normalize LIN6 expression for differences in the efficiency of cDNA synthesis, transcript levels of the constitutively expressed elongation factor 1-
of Lycopersicon esculentum (GenBank accession no. X14449
[GenBank]
) were measured using the following primers and temperature: forward primer, 5'-ACC ACG AAG CTC TCC AGG AG-3'; reverse primer, 5'-CAT TGA ACC CAA CAT TGT CAC C-3'; 60 °C. The efficiency of each primer pair was in the range of 0.95–1.0. Real-time PCR was done using the Mx 3005P QPCR system (Stratagene, La Jolla, CA, USA) with the following protocol: denaturation (95 °C for 10 min), amplification (40 cycles of 95 °C for 30 s, primer-specific annealing temperature for 1 min, and 72 °C for 30 s), and melting curve (95 °C for 1 min, 60 °C for 30 s, heating up to 95 °C with a heating rate of 0.1 °C s–1).
Data were evaluated with the MxPro software (Stratagene). To correct for well-to-well fluorescent fluctuations, normalization of the SYBR Green–dsDNA complex signal to the passive reference dye ROX, which is included in the SYBR Green PCR Mastermix, was performed. Relative LIN6 expression levels were calculated by the comparative Ct method including normalization to the constitutively expressed gene and to a control sample.
In situ stainings
To localize invertase activity in mycorrhizal tomato roots, root pieces of 3 mm were fixed, and stained as described previously (Schaarschmidt et al., 2004) using 140 µm thick cross-sections prepared with a vibrating blade microtome (VT 1000 S, Leica Microsystems, Wetzlar, Germany). Staining for invertase activity was performed with 38 mM sodium phosphate (pH 6.0), 25 U of glucose oxidase (Fluka), 0.024% (w/v) nitroblue tetrazolium (NBT), 0.014% (w/v) phenazine methosulphate, and 1% (w/v) sucrose overnight at 37 °C in the dark. Sucrose was omitted in control reactions.
Localization of the GUS protein in roots of LIN6::uidA tobacco plants growing in the split root system was carried out by staining as described by Blume and Grierson (1997). After fixation, root parts were incubated with staining solution overnight at 37 °C. Cross-sections of stained roots were cut by hand using a razor blade or, after embedding in polyethylene glycol (PEG) (Hause et al., 1996), with a microtome (HM 355, Microm International, Walldorf, Germany).
Micrographs were taken using a Zeiss ‘Axioplan’ microscope (Zeiss, Jena, Germany) equipped with a video camera (Fujix Digital Camera HC-300Z, Fuji Photo Film, Tokyo, Japan) and were processed through Photoshop 7.0 (Adobe Systems, San Jose, CA, USA).
In situ hybridization
For embedding in paraffin, root pieces of 3 mm were fixed in 4% (w/v) paraformaldehyde, 0.1% (v/v) Triton X-100 in phosphate-buffered saline (PBS) for 2 h at room temperature. After washing, root pieces were dehydrated in a graded ethanol series. Ethanol was replaced stepwise with Rotihistol (Carl Roth, Karlsruhe, Germany) and samples were then stepwise infiltrated with Paraplast (Sigma-Aldrich, Steinheim, Germany) at 60 °C. Embedded roots were cut using a microtome. For in situ hybridization, longitudinal sections and cross-sections of 8 µm thickness were mounted on slides coated with poly-L-lysine (Sigma-Aldrich). Sections were deparaffinized with Roticlear (Carl Roth) and rehydrated. After 10 min equilibration in 10 mM TRIS–HCl (pH 8.0), sections were incubated with 10 µg ml–1 proteinase K (Sigma-Aldrich) in 0.05 M TRIS–HCl (pH 7.5) and 5 mM EDTA for 30 min at 37 °C. After washing in 10 mM TRIS–HCl (pH 8.0), sections were blocked with 1% (w/v) BSA and 2 mg ml–1 glycine in the same buffer for 30 min (at room temperature). Sections were washed in 10 mM TRIS–HCl (pH 8.0), equilibrated in 0.1 M triethanolamine (pH 8.0), and then acetylated for 10 min with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0). After washing, dehydration with a graded ethanol series, and air-drying, hybridization was carried out in a humid box at 45 °C overnight. Hybridization solution consisted of 50% (v/v) formamide, 5x SSC, 0.5% (w/v) SDS, 5x Denhardt's solution, 2 mg ml–1 tRNA, and 200 U ml–1 RNase inhibitor containing 600 ng per slide of denaturated digoxigenin (DIG)-labelled sense or antisense RNA (DIG RNA labelling Kit, Roche Diagnostics, Mannheim, Germany). Slides were washed at 45 °C with 50% (v/v) formamide in 4x SSC for 10 min, afterwards with 4x SSC (10 min) and 0.2x SSC (5 min). After equilibration in STE [10 mM TRIS–HCl (pH 7.5), 1 mM EDTA and 0.5 M NaCl] at room temperature, the sections were treated with 25 µg ml–1 RNase A in STE for 20 min at 37 °C followed by washing steps of 5 min each in STE at room temperature and 0.2x SSC at 45 °C. After a short equilibration in TBS [0.1 M TRIS–HCl (pH 7.5), 0.15 M NaCl] and blocking of sections with 1% blocking reagent (Roche Diagnostics) in TBS for 30 min at room temperature, immunological detection of DIG-labelled RNA hybrids was performed using a 1:2000 diluted anti-DIG-fab fragment conjugated with alkaline phosphatase (Roche Diagnostics) according to the supplier's protocol. The colorimetric reaction was performed in a humidified box overnight at 37 °C with detection buffer [0.1 M TRIS–HCl (pH 9.5), 0.1 M NaCl, and 50 mM MgCl2] containing 0.4 M nitroblue tetazolium (NBT), 0.5 M 5-bromo-4-chloro-3-indolyl-phosphate (BCIP), and 10 mM Levamisol (Sigma-Aldrich). The reaction was stopped by washing the sections in TE [10 mM TRIS–HCl (pH 8.0) and 1 mM EDTA].
Micrographs were taken and processed as described for in situ stainings.
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Induction of invertase activity upon mycorrhization
To analyse the role of invertases in the AM, induction of invertase activity in G. intraradices-colonized wild-type tomato roots was investigated. Invertase in situ stainings revealed a high invertase activity in mycorrhizal roots (Fig. 1). Root cross-sections incubated with staining solution containing sucrose as substrate showed a strong staining in arbusculated cells and near intercellular hyphae, as well as in the root central core (Fig. 1A, C). Control reactions where sucrose was omitted (Fig. 1B) and non-mycorrhizal roots (not shown) did not exhibit any staining. To clarify which types of plant invertases are induced upon mycorrhization, activity assays were performed using the soluble protein fraction for analysis of alkaline and acid intracellular invertases and the cell wall-bound protein fraction for analysis of apoplastic invertases. Using the respective optimal pH value, this approach allows a distinction between all three types of plant invertases (Wright et al., 1998). Unfortunately, it was not possible to detect any increase in root invertase activity upon mycorrhization compared with non-mycorrhizal tomato roots (Fig. 2).
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LIN6 transcript levels in mycorrhizal versus wounded tomato roots
To analyse the invertase induction upon mycorrhization in a more sensitive way, expression studies were carried out concentrating on the apoplastic tomato invertase isoenzyme LIN6.
In a time-course analysis, over the first 5 weeks after inoculation, non-mycorrhizal and mycorrhizal roots showed a similar decrease of relative LIN6 transcript levels analysed by comparative real-time RT–PCR (Fig. 3A). After reaching a nearly constant low level, G. intraradices-colonized roots showed a significant accumulation of LIN6 mRNA 10 weeks after inoculation compared with uninoculated roots. This 2.8-fold transcript accumulation, however, could be detected after reaching the maximal mycorrhization rate 8 weeks after inoculation.
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Using higher inoculum concentrations, LIN6 induction could be observed earlier and at lower degrees of mycorrhization (Fig. 3B). Tomato plants inoculated with an inoculum concentration of at least 20% (v/v) showed only 5 weeks later an ~1.8-fold increase in LIN6 transcripts compared with non-mycorrhizal control plants. The degree of mycorrhization ranged from 25% to nearly 50% depending on the inoculum concentration. However, the LIN6 transcript levels did not increase further with increasing mycorrhization. Plants grown in 10% (v/v) inoculum did not show a significant accumulation of LIN6 mRNA at this time point, as could already be observed in the mycorrhization kinetics described above (Fig. 3A). In parallel, roots of non-mycorrhizal plants were wounded by squeezing and incubated for up to 10 h. In roots, LIN6 transcripts showed a rapid and strong accumulation, with a 12-fold increase 5 h after wounding (Fig. 3C), whereas leaves exhibited only a weakly enhanced LIN6 transcript level up to 10 h after wounding (data not shown).
Localization of LIN6 transcripts and promoter activity in mycorrhizal roots
To localize the accumulating LIN6 transcripts in the mycorrhizal root tissue, in situ hybridizations were performed using G. intraradices-colonized wild-type L. esculentum cv. Moneymaker and cv. Microtom plants (Fig. 4A–D). Upon mycorrhization, increased LIN6 transcripts occurred in the root cortex in arbusculated cells and near fungal hyphae and in the central core (Fig. 4A, C). Hybridization with a sense probe did not exhibit specific labelling (Fig. 4B, D).
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To analyse LIN6 promoter activity, transgenic N. tabacum plants were generated expressing the gene for the marker protein GUS controlled by the LIN6 promoter. LIN6::uidA tobacco plants were inoculated with G. intraradices in the split root system. Using this system, examination of tissues in the same plant clearly showed that the increased promoter activity of LIN6 was highly restricted to colonized roots (Fig. 4E–K). Mycorrhizal root parts exhibited a clear GUS staining only 7 weeks after inoculation, whereas the non-mycorrhizal roots showed only a few small stained regions (Fig. 4E, I). Cross-sections of these mycorrhizal roots and of roots harvested 11 weeks after inoculation clearly revealed an increased promoter activity in G. intraradices-colonized cells and in the root central core (Fig. 4F–H). Most non-mycorrhizal roots did not exhibit any GUS staining, except in some cases where, in a few short root segments, a faint staining in the central cylinder occurred (Fig. 4J, K).
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Discussion |
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Apoplastic cleavage of sucrose by a plant invertase is required for all interactions with hexose-using biotrophic partners missing such enzyme activity. It has been shown that AM fungi are able to take up and metabolize hexoses, mainly glucose (Solaiman and Saito, 1997; Pfeffer et al., 1999). Until now, it could not be shown, however, whether AM fungi possess sucrose-cleaving enzymes permitting them to use sucrose as a carbon source. Isolated intraradical hyphae preferentially take up glucose, and have only limited uptake of fructose and sucrose, both 2–3 times slower than glucose, as shown by a radiorespirometric assay (Solaiman and Saito, 1997). Analysis of enzyme activities of isolated intraradical hyphae and germinated spores revealed differences between symbiotic and non-symbiotic conditions, for example, for hexokinase activity, but no data exist for sucrose-hydrolysing enzymes (Saito, 1995). As shown for axenically cultured ectomycorrhizal fungi, these fungi do not exhibit apoplastic invertase activity and cannot consume sucrose (Salzer and Hager, 1991). The incapacity for sucrose uptake was also revealed for isolated protoplasts from Amanita muscaria, which were instead shown to be able to take up hexoses with a strong preference for glucose (Chen and Hampp, 1993). Although ectomycorrhizal fungi are suggested to require host apoplastic invertase, such increased activity levels could not be detected (Schaeffer et al., 1995).
In this study, induction of the apoplastic tomato invertase LIN6 was shown upon colonization with the AM fungus G. intraradices. Using sensitive methods such as comparative real-time RT–PCR and in situ hybridization, increased LIN6 transcript levels could be detected, which were found to be localized in arbusculated cells and near hyphae by in situ techniques. In addition, using transgenic LIN6::uidA tobacco plants, promoter activation in cells containing or near fungal structures could be visualized. Moreover, increased LIN6 mRNA levels and promoter activity were found in the central core of mycorrhizal roots, which are characterized by a higher sink function. Non-mycorrhizal roots showed no or only less GUS staining, which was restricted to the central core, indicating promoter activity in such cells. These points confirm the postulated role of LIN6 in phloem unloading (Godt and Roitsch, 1997). To our knowledge, increased transcript levels of apoplastic invertases in AM-colonized roots has not been shown previously, in contrast to those of other sucrose-cleaving enzymes, which in some studies were found to be induced (Blee and Anderson, 2002; Hohnjec et al., 2003; Ravnskov et al., 2003).
Although extracellular invertases are suggested to play an important role in supplying sink organs with carbohydrates, it seemed to be hard to detect increased activity or transcript levels. Only a few previous studies revealed enhanced acid invertase activity in obligate biotrophic fungi–plant interactions including parasitic interactions, for example, reported for powdery mildew (Storr and Hall, 1992; Scholes et al., 1994; Fotopoulos et al., 2003) and rust (Long et al., 1975; Krishnan and Pueppke, 1988; Tetlow and Farrar, 1992) as well as mutualistic interactions (Wright et al., 1998). This could be caused by technical problems, as increased invertase activity levels could be detected near AM fungal structures by in situ activity staining but not by enzyme assays using extracted protein fractions. Due to the highly localized induction of gene expression in infected cells, this effect might be due to a dilution by extracting the complete root system of mycorrhizal plants, which always contains a high proportion of non-mycorrhizal cells. A similar effect was already described by Blee and Anderson (2002), who could detect increased transcript levels of genes encoding vacuolar invertase and sucrose synthase in arbusculated cells by in situ hybridization but not via northern analysis of whole root tissues. Furthermore, the authors failed to detect increased enzyme activities by extracting the whole root. This might be based on the fact that the analysis of transcript levels offers more sensitive methods compared with enzyme activity assays. Moreover, in situ methods provide a powerful tool for sensitive tissue- or cell-specific analysis. In addition, in situ invertase activity staining would also encompass a potential post-translational repression of the enzyme activity by proteinaceous (Rausch and Greiner, 2004) or chemical (Höke and Dräger, 2004) invertase inhibitors present in the tissue, showing by this the biologically effective, local invertase activity.
In the present study, increased LIN6 transcripts could be detected at late stages of mycorrhization in a fully established symbiosis (10 and 11 weeks after inoculation) or, if plants were inoculated with a higher density of fungal structures, at earlier stages before reaching the maximal mycorrhization rate (5 weeks after inoculation). This suggests a threshold of fungal demand for hexose to induce LIN6 transcription. Long-term experiments revealed increasing activity levels of extracellular invertases over 70 d in mycorrhizal clover (Wright et al., 1998). By contrast, cytosolic invertase activity was elevated, but remained constant in comparison with levels in non-mycorrhizal roots. Increased enzyme activities of sucrose synthase and vacuolar invertases in these roots were detected particularly in the early stages of mycorrhization, though increased vacuolar invertase activity seemed to be caused by parallel nodulation of mycorrhizal clover roots. In G. max, apoplastic and vacuolar invertases showed no enhanced activity due to AM up to an experimental duration of 40 d, while cytosolic invertase activity was already enhanced (Schubert et al., 2003). Although these studies do not implicate increased vacuolar invertase activity upon mycorrhization, increased transcript levels of a gene coding for a vacuolar invertase suggested an important function for this type of plant invertase in the AM interaction (Blee and Anderson, 1998, 2002). Thus, distinct roles for the different types of plant invertases during the process of AM are suggested. Cytosolic and vacuolar invertases might be more important in metabolizing intracellular sucrose at the beginning of mycorrhization. In this case, the intracellular hydrolysis of sucrose would maintain a gradient for symplastic influx of sucrose into colonized cells and establish a gradient for the efflux of hexoses to the apoplast (Blee and Anderson, 1998). By contrast, higher activities of extracellular invertases seemed to be particularly involved in stages requiring high carbohydrate levels like a fully established AM to maintain the increasing sink strength by phloem unloading and to provide apoplastic hexoses. Such higher activities might be reached by acidification of the arbuscular interface, enhancing the activity of existing extracellular invertases (Guttenberger, 2000), or by induction of protein biosynthesis which might be indicated by transcript accumulation as shown for LIN6. Two such phases of invertase induction could already be seen in wounded tomato leaves (Godt and Roitsch, 1997). After wounding, transcripts of a vacuolar invertase accumulated transiently between 1 h and 24 h after wounding, whereas LIN6 transcripts could be detected from 24 h onwards. This suggests establishment of sink strength in wounded leaves after mobilization of local reserves.
LIN6 was found to be sensitive to different stress-related stimuli such as wounding (Godt and Roitsch, 1997) or the application of brassinosteroids (Goetz et al., 2000) and methyl jasmonate (Thoma et al., 2003). By wounding of tomato roots, a rapid, up to 12-fold increase in LIN6 mRNA levels of the roots was observed, whereas G. intraradices inoculation led to a 2- or 3-fold increase in LIN6 transcript level, measured in whole root tissue. Thus, in tomato roots, LIN6 seemed to be highly susceptible to external stress. In return, a severe modulation of source–sink activities and strong induction of extracellular invertases could result in an activation of defence-related mechanisms of the plant via sugar-mediated gene expression (Rolland et al., 2002; Roitsch et al., 2003), which would interfere with the mutualistic interaction. Therefore, the dual roles of extracellular invertase in stress responses and assimilate partitioning (Roitsch et al., 2003) need to be balanced. The susceptibility of roots to stress stimuli and the linkage between stress-induced invertase induction and activation of defence mechanisms requires a co-ordinated regulation and fine-tuned increase of hexose-providing enzymes, such as the observed moderate induction of LIN6 in the present study, by AM.
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Acknowledgements |
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We greatly acknowledge Professor Dr Dieter Strack and Dr Margaret Rice for critically reading the manuscript.
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AM, arbuscular mycorrhiza; GUS, ß-glucuronidase; LIN, Lycopersicon invertase; RT–PCR, reverse transcription–polymerase chain reaction.
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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