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Keywords:

  • abscisic acid;
  • apoplastic invertase;
  • arbuscular mycorrhiza;
  • defense response;
  • metabolite profiling;
  • Nicotiana tabacum (tobacco)

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The effect of constitutive invertase overexpression on the arbuscular mycorrhiza (AM) is shown. The analysis of the enhanced potential for sucrose cleavage was performed with a heterozygous line of Nicotiana tabacum 35S::cwINV expressing a chimeric gene encoding apoplast-located yeast-derived invertase with the CaMV35S promoter. Despite the 35S promoter, roots of the transgenic plants showed no or only minor effects on invertase activity whereas the activity in leaves was increased at different levels. Plants with strongly elevated leaf invertase activity, which exhibited a strong accumulation of hexoses in source leaves, showed pronounced phenotypical effects like stunted growth and chlorosis, and an undersupply of the root with carbon. Moreover, transcripts of PR (pathogenesis related) genes accumulated in the leaves. In these plants, mycorrhization was reduced. Surprisingly, plants with slightly increased leaf invertase activity showed a stimulation of mycorrhization, particularly 3 weeks after inoculation. Compared with wild-type, a higher degree of mycorrhization accompanied by a higher density of all fungal structures and a higher level of Glomus intraradices-specific rRNA was detected. Those transgenic plants showed no accumulation of hexoses in the source leaves, minor phenotypical effects and no increased PR gene transcript accumulation. The roots had even lower levels of phenolic compounds (chlorogenic acid and scopolin), amines (such as tyramine, dopamine, octopamine and nicotine) and some amino acids (including 5-amino-valeric acid and 4-amino-butyric acid), as well as an increased abscisic acid content compared with wild-type. Minor metabolic changes were found in the leaves of these plants. The changes in metabolism and defense status of the plant and their putative role in the formation of an AM symbiosis are discussed.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

In autotrophic plants, sucrose represents the major carbon transport form from photosynthetically active source leaves to heterotrophic sink tissues. Efficient carbon allocation and provision of utilizable carbohydrates is key for successful plant growth and development. Apoplastic invertases, also termed cell wall-bound or extracellular invertases, are known as important regulators in such processes. By phloem unloading they can establish and maintain increasing sink strength. Hydrolysis of sucrose by extracellular invertases at the site of phloem unloading drives the long-distance carbon transport to sink organs powered by differences in osmotic potentials. The cleavage products glucose and fructose can directly serve as carbon and energy sources for the plant cells. Next to the direct feeding of the plant cell, sucrose and hexoses can act as metabolic signals influencing plant gene expression and development (for reviews, see Koch, 1996; Rolland et al., 2006). In general, hexoses favor cell division and expansion, whereas sucrose favors differentiation and maturation (for review, see Koch, 2004). Moreover, sugars are important signals in the activation of defense-related mechanisms (for reviews, see Rolland et al., 2002, 2006; Salzer et al., 2000).

In addition to apoplastic invertases, sucrose can be hydrolyzed in plants by the action of vacuolar or cytosolic invertases and cytosolic sucrose synthase. Whereas the latter cleaves sucrose reversibly into UDP-glucose and fructose, all invertases hydrolyze sucrose to glucose and fructose, which are both, in contrast to UDP-glucose, known to trigger hexose signal function. Similar to apoplastic invertases, the vacuolar invertases are N-glycosylated-β-fructofuranosidases with an acidic pH optima. Specific physiological functions of the different subcellularly located sucrose-cleaving enzymes have been demonstrated for plant developmental processes (for reviews, see Sturm and Tang, 1999; Koch, 2004; Roitsch and González, 2004). Regarding apoplastic invertases, the specific expression pattern of different isoenzymes in a development- and organ-specific manner allows a fine-tuned response to certain stimuli, and indicates the important regulatory function of these enzymes. For example, expression analysis of the extracellular tomato invertases showed that three of four isoenzymes (LIN5, LIN6 and LIN7) are expressed in flowers – but in distinct developmental stages and/or different flower organs (Godt and Roitsch, 1997; Fridman and Zamir, 2003; Proels et al., 2003). Antisense suppression of the LIN7 tobacco homolog NIN88 resulted in male sterility (Goetz et al., 2001), supporting the crucial role of one apoplastic invertase isoenzyme in pollen development.

Apoplastic invertases are known to respond to a whole set of stimuli including sugars, phytohormones, and abiotic and biotic stressors. For example, induction by glucose and the growth-promoting phytohormone zeatin was found for LIN6 (Godt and Roitsch, 1997), whereas vacuolar invertase and sucrose synthase showed no response. Moreover, LIN6 was strongly induced by stress-related stimuli such as polygalacturonic acid, mimicking pathogen infection, brassinosteroids, methyl jasmonate and wounding of leaves or roots (Godt and Roitsch, 1997; Goetz et al., 2000; Thoma et al., 2003; Schaarschmidt et al., 2006). An activation by gibberellic acid, auxin and abscisic acid (ABA) was found for the LIN5 promoter (Proels et al., 2003). The tapetum- and pollen-specific LIN7 was found to be at least gibberellic acid inducible (Proels et al., 2006). In addition to wounding, apoplastic invertases can be also induced by other abiotic stresses like drought or salt stress (Roitsch et al., 2003; Ji et al., 2005). Upon biotic stress, higher transcript or activity levels of extracellular invertases were detected, e.g. in potato virus Y (PVY) infected tobacco (Herbers et al., 2000), and upon infection with several pathogenic fungi, such as rice blast (Magnaporthe grisea), rust (Pucchinia hordei) and powdery mildew (Erysiphe cichoracearum) (Tetlow and Farrar, 1992; Fotopoulos et al., 2003; Cho et al., 2005). In mutualistic plant–fungus interactions, like ectomycorrhiza and arbuscular mycorrhiza (AM), apoplastic invertases are induced to support the increased sink strength caused by the feeding of the symbiotic partner (Salzer and Hager, 1991; Wright et al., 1998, 2000; Schaarschmidt et al., 2006).

In response to this regulation of apoplastic invertase by certain stimuli, invertases release hexoses as signals for several, inter alia stress-related mechanisms, including phytohormone accumulation, expression of defense genes and a feedback induction of invertases. Such hexose-triggered transcript accumulation has been shown, e.g. for the defense gene PAL and the cell wall invertase gene CIN1, both strongly expressed in glucose-treated Chenopodium rubrum cell culture (Ehness et al., 1997). Thus, extracellular invertases belong to a complex network of regulatory elements amplifying the initial signal (for reviews, see Leon and Sheen, 2003; Roitsch et al., 2003).

Overexpression of genes coding for extracellular-located invertase can affect hormonally regulated developmental processes, and increase the tolerance against abiotic and biotic stresses. In this respect, senescence-induced invertase-overexpressing tobacco leaves exhibited a cytokinin-mediated delay of leaf senescence (Balibrea Lara et al., 2004). The constitutive overexpression results in an improved salt-stress tolerance (Fukushima et al., 2001), accumulation of PR gene transcripts, increased salicylate levels and accumulation of phenolic compounds accompanied by increased resistance against PVY (Herbers et al., 1996; Baumert et al., 2001). The effect of constitutive overexpression of apoplastic invertase on mutualistic interactions, including the AM fungi, is, however, unknown. The AM fungi, forming a mutualistic symbiosis with most land plants (for review, see Hause and Fester, 2005), are dependent on the sucrose-cleaving enzymes of their host, as they are only able to take up hexoses from the plant apoplast within their intraradical mycelium (Pfeffer et al., 1999; Douds et al., 2000). No uptake could be observed by extraradically grown mycelium. Therefore, plant apoplastic invertases are suggested to be involved in providing the fungus with hexoses. Moreover, they might have a key function in maintaining increasing sink strength by phloem unloading. This is supported by the fact that in AM roots, both transcript accumulation of a gene coding for an apoplastic invertase and activity of apoplastic invertases are enhanced near fungal structures and in the phloem cells (Schaarschmidt et al., 2006). Moreover, reduced apoplastic invertase activity accomplished by the root-specific overexpression of a proteinacous inhibitor leads to a diminished mycorrhization (Schaarschmidt et al., 2007). Thus, upon constitutive invertase overexpression, the AM fungus might benefit from a higher availability of carbohydrates, and the mycorrhization could be enhanced. On the other hand, activation of defense reactions as a result of enhanced apoplastic invertase activity might interfere with the mutualistic interaction, and could lead to diminished mycorrhization.

Hence, in the present study the effect of constitutive overexpression of apoplastic invertases on formation of AM was analyzed. A heterozygous line of Nicotiana tabacum (NT) 35S::cwINV plants (von Schaewen et al., 1990) was used, expressing a chimeric invertase gene with the CaMV35S promoter. The chimeric invertase gene consists of the signal peptide of potato proteinase inhibitor II and the coding sequence of the yeast invertase gene suc2 and encodes an N-glycosylated invertase, which is efficiently secreted to the apoplast (von Schaewen et al., 1990). Heterozygous NT35S::cwINV plants were characterized by their invertase activity, sugar levels and defense status, and were analyzed in view of their colonization with the AM fungus Glomus intraradices. Surprisingly, either a stimulation or a repression in the fungal colonization of the root was observed, depending on the leaf invertase activity. Thus, a regulatory function of apoplastic invertases on the whole-plant level is suggested.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Relations between invertase activity, contents of soluble sugars, phenotypical effects and mycorrhization

Plants of a heterozygous NT 35S::cwINV line showed a broad spectrum in their individual invertase activity and their soluble sugar levels in leaves. Here, the data of G. intraradices-inoculated plants of two independent experiments are presented, which differed strongly in the overall mycorrhization degree (Figure 1; see also Figure S1). Using this experimental set-up, alterations of parameters between wild-type and transgenic plants can be referred to the effect of increased apoplastic invertase activity, rather than to effects caused by the mycorrhization itself. The non-mycorrhizal plants showed a similar spectrum in their invertase activity and the same behavior in their sugar levels as mycorrhizal plants (in Figure S2 the data of one selected time-point of each experiment is given).

image

Figure 1.  Invertase activity, ratio of soluble sugars and mycorrhization in invertase-overexpressing plants. Two independent experiments (I and II) with different overall mycorrhization by Glomus intraradices were performed to exclude effects resulting from the colonization rate. (a) Invertase activity in roots and leaves of wild-type and NT 35S::cwINV plants. (b) Ratio of the sum of glucose and fructose to the sucrose content of these plants. (c) Degree of mycorrhization. (d) Relative levels of G. intraradices-specific rRNA. Levels of fungal rRNA in wild-type roots, 3 weeks after inoculation, were set to 1. Six-week-old plants were inoculated with G. intraradices and harvested in experiment I after 3 and 4.5 weeks, and in experiment II after 3 and 6 weeks. Heterozygous plants expressing the chimeric invertase gene were grouped by their leaf invertase activity as follows: ‘35S::cwINV; A’, leaf invertase activity up to 50 pkat mg−1 (protein), ‘35S::cwINV; B’, between 50 and 100 pkat mg−1 (protein); ‘35S::cwINV; C’, between 100 and 200 pkat mg−1 (protein); ‘35S::cwINV; D’: over 200 pkat mg−1 (protein). Data are given as mean values +SD of between three and eight plants per group and are tested with a one-way anova followed by Tukey’s HSD test (a, b, d); P < 0.05. Means sharing the same letters are not significantly different.

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Leaf invertase activity was found to be increased from about twofold up to more than 25-fold compared with wild-type (Figure 1a). Despite expression of the chimeric invertase gene under control of the 35S promoter, however, root invertase activity was only slightly enhanced (up to threefold) or not affected at all. Hence, transgenic plants were divided by their leaf invertase activity into four groups: (A) up to 50 pkat mg−1 (protein), (B) in the range from 50 to 100 pkat mg−1 (protein), (C) in the range from 100 to 200 pkat mg−1 (protein) and (D) more than 200 pkat mg−1 (protein). Leaf invertase activity of wild-type plants averaged at 12 pkat mg−1 (protein). It became obvious that slightly increased invertase activities in NT 35S::cwINV leaves (from two- to fourfold) are mainly observed at earlier developmental stages (3 weeks after inoculation). At later stages (6 weeks after inoculation) the invertase activity was generally higher, possibly because of an accumulation of invertase protein over time. In some plants an about 25-fold higher invertase activity than in the wild-type was found (Figure 1a).

According to their invertase activity, NT 35S::cwINV plants showed, particularly at later developmental stages (4.5 and 6 weeks after inoculation), markedly elevated hexose (glc + frc) contents (Figure S1a) and hexose: sucrose ratios in the leaves (Figure 1b). The sucrose content in leaves was less affected (Figure S1b). In transgenic plants with slightly increased leaf invertase activity (group A) levels of soluble sugars were not affected in the leaves or even tended downwards (see experiment I, 3 weeks after inoculation). Roots of all plants showed constant or reduced sugar levels and constant hexose: sucrose ratios compared with wild-type roots (Figure S1a,b and Figure 1b).

Interestingly, we found a correlation between the leaf invertase activity and the degree of mycorrhization in NT 35S::cwINV plants. At early stages (3 weeks after inoculation), plants of both experiments with slightly (from two- to fourfold) increased invertase activity in leaves showed a higher degree of mycorrhization and increased levels of G. intraradices-specific rRNA (Figure 1c,d; group A). Furthermore, we could detect a higher density of fungal structures compared with wild-type plants (Figure 2b,c; see also Figure S3a–d). In contrast, mycorrhization and fungal rRNA levels decreased with increasing leaf invertase activity over time, resulting in repressed mycorrhization (Figure 1c,d; groups B–D), both relatively to plants with enhanced mycorrhization at early time-points and to wild-type plants at later developmental stages. This points to a long-term effect of accumulating invertase protein/activity on the development of AM. In addition to a decreased degree of mycorrhization, staining of fungal structures revealed less formation of vesicles, representing the fungal storage organs, than in wild-type roots (Figure 2e,f). Moreover, in contrast to transgenic plants of group A that showed well-developed arbuscules, in plants with strongly increased leaf invertase activity particularly small arbuscules were found (Figure S3e). In some root segments only intercellular hyphae could be detected. A similar colonization pattern with less formation of fungal vesicles was also found in transgenic NT rolC::PPa plants with an undersupply of the root (Figure S3f; Schaarschmidt et al., 2007). In these plants, the phloem-specific expression of the pyrophospatase gene ppa from Escherichia coli inhibits the inorganic pyrophosphate (PPi)-dependent uptake of sucrose into the phloem cells resulting in sugar accumulation in source leaves and an undersupply of the sink organs (Lerchl et al., 1995).

image

Figure 2.  Phenotypes of invertase-overexpressing plants and formation of mycorrhizal structures. (a, d) Phenotypes of wild-type and representative 35S::cwINV tobacco plants of group A [leaf invertase activity below 50 pkat mg−1 (protein)] and of group C [leaf invertase activity more than 100 pkat mg−1 (protein)] 3 weeks (a) and 6 weeks (d) after inoculation with Glomus intraradices, respectively. All plants were inoculated 6 weeks after sowing. (b, c, e, f) Ink-stained fungal structures in roots of wild-type (b, e) and invertase-overexpressing plants (c, f) harvested 3 weeks (b, c) and 6 weeks (e, f) after inoculation.

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Plant phenotype and PR gene expression in invertase-overexpressing plants

The slightly increased leaf invertase activity in 35S::cwINV plants led to marginal alterations of the plant phenotype (Figure 2a). Only minor growth reduction and leaf chlorosis could be observed. The content of total chlorophyll was not significantly changed compared with wild-type plants (Figure 3a). In contrast, plants with strongly elevated leaf invertase activity showed a pronounced phenotype with stunted growth and chlorosis of leaves (Figure 2d). Total chlorophyll content as well as stem length and leaf size of NT 35S::cwINV decreased with increasing invertase activity compared with wild-type, especially at later developmental stages (Figure 3a,b). The number of leaves was less affected, but also decreased in comparison with wild-type plants 6 weeks after inoculation (Figure 3b). These phenotypes were similar in non-mycorrhizal and mycorrhizal NT 35S::cwINV plants (Figure S2).

image

Figure 3.  Chlorophyll content and growth parameters of wild-type and invertase overexpressing plants. (a) Content of total chlorophyll of wild-type and NT 35S::cwINV plants of two independent experiments (experiment I and II; see Figure 1). (b) Length of stem, number of fully-developed leaves and length of the largest leaf, used as indicator for leaf size, of wild-type and NT 35S::cwINV plants 6 weeks after inoculation. All plants were inoculated with Glomus intraradices 6 weeks after sowing. The plants were grouped by their leaf invertase activity as described in Figure 1. Data are presented as mean + SD of between three and eight plants per group. Different letters designate statistically different values (anova with Tukey’s HSD test; P < 0.05).

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As described previously, constitutive expression of apoplast-located yeast invertase can lead to an enhanced defense status of the plant (Herbers et al., 1996, 2000). To test whether defense mechanisms are induced in heterozygous NT 35S::cwINV plants, gene expression analysis of PAR1, PR-Q and PR-1b, which were found to accumulate in leaves of homozygous NT 35s::cwINV plants (Herbers et al., 1996), was performed by semi-quantitative RT-PCR.

In invertase-overexpressing plants with slightly enhanced leaf invertase activity and increased mycorrhization (group A, 3 weeks after inoculation), transcripts of the analyzed PR genes did not accumulate to higher levels than in wild-type (Figure 4). In leaves with high invertase activity (groups B–D), however, PAR1, PR-Q and PR-1b transcript levels accumulated to higher levels compared with wild-type leaves. In contrast, increased transcript accumulation of PR genes was not detected in roots. There, PR gene transcript levels were not changed (PAR1 and PR-1b) or seemed even to be lower than in the wild-type (PR-Q). Thus, in the analyzed plants induction of PR gene expression by elevated leaf invertase activity was found to be local instead of systemic.

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Figure 4.  Expression analysis of PR genes in wild-type and invertase-overexpressing tobacco. Transcript levels of PAR1, PR-Q and PR-1b were analyzed in pooled root and leaf samples of at least three mycorrhizal wild-type and NT 35S::cwINV plants via RT-PCR. Plants were inoculated with Glomus intraradices, harvested at two time-points and grouped by their leaf invertase activity as described in Figure 1. The quantity of template used for RT-PCR is given in the figure.

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Hormone and metabolite status of invertase-overexpressing plants with stimulated mycorrhization

To analyze metabolic alterations possibly leading to the stimulated mycorrhization in NT 35S::cwINV plants with slightly increased leaf invertase activity compared with wild-type plants, steady-state analyses of phytohormones and polar metabolites were performed. Roots and leaves of wild-type and NT 35S::cwINV plants of group A of both experiments, harvested 3 weeks after inoculation with G. intraradices, were investigated. To exclude effects caused by different mycorrhization levels of wild-type and transgenic plants, both experiments were designed with different overall colonization rates. Effects exclusively resulting from differences in the mycorrhization would not only lead to changed ratios between transgenic and wild-type plants but also to appropriate changes in the absolute values between both experiments. This was mostly not observed. In both experiments (experiment I and II), significantly (1.8- and 2.7-fold) increased ABA levels were found in roots of invertase-overexpressing plants compared with wild-type roots (Figure 5). In leaves, the ABA content was less affected showing a significant increase (1.8-fold) in only one experiment. Contents of total, free and conjugated indole-3-acetic acid (IAA) showed a non-reproducible increase in roots of the analyzed NT 35S::cwINV plants compared with wild-type roots (Figure S4; see experiment II). IAA contents in leaves were not significantly affected by increased invertase activity.

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Figure 5.  Abscisic acid (ABA) content in wild-type plants and NT 35S::cwINV plants with increased mycorrhization. The ABA content in roots and leaves of mycorrhizal wild-type and 35S::cwINV tobacco plants of group A with slightly elevated leaf invertase activity [below 50 pkat mg−1 (protein)] 3 weeks after inoculation with Glomus intraradices is shown. Data of two independent experiments (experiment I and II; see Figure 1) are given each as mean value +SD of between three and five plants. In total eight wild-type and eight transgenic plants were analyzed. Data of the transgenic lines were compared with the wild-type by the Student’s t-test; *P < 0.05, **P < 0.002.

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Analysis of polar compounds revealed a clear separation between the metabolic steady-state of roots and leaves, as well as more pronounced differentiations between wild-type and 35S::cwINV plants than between both experiments (Figure 6a). The identified compounds showing a change in pool size for roots and/or leaves of all wild-type and 35S::cwINV plants of both experiments (experiments I and II) are listed in Table 1. In Table S1 the subset of all identified mass fragments is given. Interestingly, more metabolic changes occurred in roots than in leaves of invertase-overexpressing plants compared with wild-type. Most of them become apparent as decreased metabolite levels.

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Figure 6.  Independent component analysis (ICA) of metabolite profiles from roots (diamonds) and leaves (circles) of Nicotiana tabacum wild-type (open symbols) and 35S::cwINV plants of group A showing increased mycorrhization (closed symbols). Two independent experiments, I (small symbols) and II (large symbols), with different overall mycorrhization degrees were performed to exclude effects caused by different mycorrhization levels as described in Figure 1. (a) Analysis of ICA scores demonstrates common differences in root and leaf metabolism (IC2) in response to slightly enhanced leaf invertase activity in the data set (Table S1). (b) Loading analysis demonstrates the contribution of metabolites to this metabolic differentiation. All detected GC-TOF-MS mass fragments are shown and the top-scoring identified metabolites are indicated: 1, carbodiimide; 2, nicotianamine; 3, erythritol; 4, glyceric acid-3-phosphate; 5, nicotine; 6, octopamine; 7, caffeoylquinic acid (chlorogenic acid); 8, dopamine; 9, glucose; 10, glycine; 11, allantoin; 12, fructose-6-phosphate; 13, phosphoric acid; 14, tryptophan; 16, scopolin.

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Table 1.   Steady-state analysis of polar metabolites from roots and leaves of Nicotiana tabacum wild-type and 35S::cwINV plants with increased mycorrhization. This table shows the identified metabolites with a changed pool size between wild-type and transgenic plants. The subset of identified metabolites and the criteria of metabolite identification are documented in Table S1. Metabolite accumulation in NT 35S::cwINV plants of group A (INV), characterized by slightly elevated leaf invertase activity [below 50 pkat mg−1 (protein)], were compared with wild-type (wt). All plants were harvested 3 weeks after inoculation with Glomus intraradices in two independent experiments (experiment I and II). Polar metabolites were analyzed by GC-TOF-MS-based metabolite profiling of methoxyaminated and trimethylsilylated metabolite analytes. Significant changes (P < 0.05) between wild-type and transgenic plants are presented in bold (wt, n = 8; INV, n = 9). dRI, difference between measured and expected retention index; FM, fragment mass; MF, matching factor; nd, not detected; nc, ratio cannot be calculated.
MetaboliteAnalyteChange of Pool Size
NameSum formulaKEGG-IDCAS-IDMPIMP-IDFM [amu] dRI [%]MF [best match]roots x-fold [INV/wt]Pleaves x-fold [INV/wt]P
  1. aRepresents the sum of two or more metabolites.

  2. bCarbodiimide is formed from the guanidinio-moiety of arginine and agmatine.

  3. cPresent in 35S::cwINV plants but below detection limit in all except one wild-type sample.

  4. dReference substance not yet available.

Organic acids
 Succinic acidC4H6O4C00042110-15-6A134001-1012470.019850.750.0101.270.111
 Itaconic acidC5H6O4C0049097-65-4A135004-1011330. 039261.290.1211.610.021
 Glutaric acid, 2-oxo-C5H6O5C00026328-50-7A158004-1011980.139131.210.0291.130.665
Amino acids
 AlanineC3H7NO2C0004156-41-7A138002-101188−0.289560.680.0321.100.387
 Alanine, beta-C3H7NO2C00099107-95-9A144001-101248−0.139040.480.0000.940.583
 Butyric acid, 4-amino-C4H9NO2C0033456-12-2A153003-101174−0.149720.640.0580.980.880
 Valeric acid, 5-amino-C5H11NO2 660-88-8A164005-101174−0.159200.490.0000.720.068
 ProlineC5H9NO2C00148147-85-3A132003-101142−0.149630.480.1240.420.017
N-Compounds
 Carbodiimide   (Agmatine, Arginine)a,bCH2N2  A100005-1011710.58Manual5.510.0008.220.003
 AdenineC5H5N5C0014773-24-5A188005-1012640.16Manual0.730.0230.970.926
 Nicotinic acidC6H5NO2C0025359-67-6A133004-1011800.169420.770.0060.960.788
 SpermidineC7H19N3C00315124-20-9A220002-101174−0.158660.890.3681.110.851
 SpermidineC7H19N3C00315124-20-9A226002-101144−0.239330.760.0360.710.127
 TyramineC8H11NOC0048351-67-2A191004-101174−0.029370.570.0040.750.029
 Tyramine, 3-methoxy-C9H13NO2C05587554-52-9A204002-101174−0.079340.600.0111.070.967
 DopamineC8H11NO2C0375851-61-6A208001-101100−0.13Manual0.470.000nd 
 OctopamineC8H11NO2 104-14-3A203001-1011740.099160.470.0000.340.016
 NicotineC10H14N2 54-11-5A139002-101840.029700.320.0190.370.094
Phenylpropanoids and phenolic compounds
 ScopoletinC10H8O4 92-61-5not assigned2340.347020.460.0011.960.243
 Quinic acid, 3-caffeoyl-, cis-C16H18O9  A299001-101255−0.129370.650.0330.900.463
 Quinic acid, 3-caffeoyl-, trans-C16H18O9C00852327-97-9A311001-101255−0.199390.500.0240.580.033
 Quinic acid, 4-caffeoyl-, trans-C16H18O9  A317001-101307−0.089560.780.3130.580.026
 Quinic acid, 5-caffeoyl-, trans-C16H18O9C03908 A319001-101307−0.089421.100.5470.570.021
Phosphates
 Phosphoric acidH3O4PC000097664-38-2A129001-101299−0.019821.950.0001.860.003
 Glyceric acid-3-phosphateC3H7O7PC00197820-11-1A181003-1012990.088249.560.0292.040.621
 Glycerol-3-phosphateC3H9O6PC0009329849-82-9A177002-101299−0.027262.320.0001.250.135
 Mannose-6-phosphateC6H13O9PC00275 A231001-101387−0.028242.490.0002.140.012
 Fructose-6-phosphateC6H13O9PC00085643-13-0A232002-101315−0.138432.430.0002.040.001
 Glucose-6-phosphateC6H13O9PC0009256-73-5A233002-101387−0.129112.800.0001.820.001
Polyhydroxy acids
 Glucuronic acidC6H10O7C001911700-90-8A193004-101160−0.058600.430.0080.910.969
 Galactaric acidC6H10O8C01807526-99-8A204001-101333−0.219071.360.035nd 
 Gluconic acidC6H12O7C00257526-95-4A200001-101333−0.23Manual0.510.0471.770.815
Polyols
 ErythritolC4H10O4C00503149-32-6A150002-101217−0.159471.980.1304.410.007
 GalactinolC12H22O11C01235 A299002-1012040.07Manual0.800.0331.090.475
Monosaccharides
 XyloseC5H10O5C0018158-86-6A165001-101217−0.08Manual0.440.0020.760.060
 RiboseC5H10O5C0835350-69-1A168002-101217−0.149350.520.0000.870.374
 Glucose, 1,6-anhydroC6H10O5 498-07-7A172001-1012040.009460.350.0001.050.693
 RhamnoseC6H12O5C005073615-41-6A172002-101117−0.298360.550.0070.790.174
 FucoseC6H12O5C010183615-37-0A173002-101117−0.299030.550.0000.930.389
 FucoseC6H12O5C010183615-37-0A175001-101117−0.24Manual0.610.0000.680.145
 FructoseC6H12O6C0009557-48-7A187002-101217−0.259690.270.0010.760.563
 FructoseC6H12O6C0009557-48-7A188004-101217−0.259830.150.0020.770.617
 GlucoseC6H12O6C0003150-99-7A189002-101160−0.23Manual0.260.0010.500.297
 GlucoseC6H12O6C0003150-99-7A191001-101205−0.27Manual0.190.0030.490.296
Trisaccharides
 RaffinosecC18H32O16C00492512-69-6A337002-101361−0.39Manualnc nc 
MSTsd
 [Benzylglucopyranoside (4TMS)]   A241003-101910.068900.480.0011.500.102
 [819; NIST108989; Scopolin (4TMS)] C01527513-44-2A318002-101264−0.079500.610.041nd 

By analyzing polar compounds via GC-MS we found lower monosaccharide contents in the roots of transgenic plants in comparison with wild-type roots (Table 1). In addition to decreased glucose and fructose levels, xylose, ribose, rhamnose and fucose were detected in lower quantities. The leaves were not significantly affected in their monosaccharide contents. This is reminiscent to non-mycorrhizal plants, which were analyzed by photometric sugar measurements and showed a similar reduction in transgenic plants in comparison with wild-type plants (Figure S2). In contrast to monosaccharides, raffinose was found predominantly in the transgenic plants (Figure S5), which also contained markedly elevated contents of polyol erythriol, sugar phosphates and some other phosphates. An enhanced nutrient supply of 35S::cwINV plants with increased mycorrhization might be indicated by on average four- and eightfold higher levels of carbodiimide in roots and leaves, respectively, and by higher levels of a novel amine. Its putative role is unknown, but it could, however, be detected in all tobacco plants and was found to be more than 100-fold increased in roots of invertase-overexpressing plants compared with wild-type, which contrasts to all identified amines (Table S1 and Figure S5).

Polyhydroxy acids and organic acids were less affected in the transgenic plants. Thus, leaves of slightly invertase-overexpressing plants showed only an enhanced content of itaconic acid, whereas their roots had lower contens of gluconic, glucuronic and succinic acids (Table 1). In addition, some amino acids or amino acid derivatives were reduced in 35S::cwINV plants. In roots, these were identified as alanine, 5-amino-valeric acid and 4-amino-butyric acid (GABA), the latter had a borderline P-value for the changed pool size of both experiments but was significantly reduced in each experiment (see also Figure S6). In leaves, proline was the only amino acid showing a reduced level. Interestingly, the transgenic plants had also, especially in roots, lower contents of defense-related metabolites, such as amines and phenolic compounds. In roots of invertase-overexpressing plants with increased mycorrhization, chlorogenic acid (caffeoylquinic acid), scopolin, scopoletin and all identified amines were reduced (Table 1 and Figure S6). The latter includes octopamine, nicotine, dopamine, tyramine and methoxytyramine; spermidin also appeared to be decreased. Moreover, reduced levels of benzylglucopyranoside occurred in roots of 35S::cwINV plants with slightly increased invertase activity. Nicotine, octopamine, dopamine, chlorogenic acid and scopolin are among the top-scoring identified metabolites contributing to the found metabolic differentiation (Figure 6b). However, the reduced levels of chlorogenic acid, octopamine and tyramine in the leaves were mainly the result of metabolic changes in one experiment only (experiment I; see Figure S6).

Thus, slightly increased invertase activity in tobacco leaves resulted in a marked change of the metabolic status of the root, with increased ABA contents and decreased levels of defense-related compounds like phenolic compounds, amines and some amino acids, whereas only minor effects in the leaves could be detected.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

AM fungi are obligate biotrophic organisms and have to be supplied with carbohydrates by the plant, most likely in the form of hexoses (Solaiman and Saito, 1997; Pfeffer et al., 1999; Douds et al., 2000). Therefore, apoplastic invertases are believed to play a crucial role in the AM symbiosis (Bago et al., 2000; Schaarschmidt et al., 2006). To address this by a functional approach, alterations in mycorrhization in response to overexpression of an apoplastic invertase were studied. As shown recently, root-specific elevated apoplastic invertase activity, achieved by expressing a chimeric gene encoding apoplast-located yeast-derived invertase with an alcohol-inducible promoter, does not affect AM in transgenic tobacco plants (Schaarschmidt et al., 2007). In contrast, root-specific expression of an acid invertase inhibitor gene resulted in reduced apoplastic invertase activity and decreased mycorrhization in roots of transgenic tobacco (Schaarschmidt et al., 2007). In the present study, the effect of constitutive overexpression of apoplastic invertase on the formation of AM was analysed. For that reason, transgenic NT 35S::cwINV plants of a heterozygous line were used, containing different copy numbers of the chimeric gene coding for apoplast-located yeast invertase, which is under the control of the CaMV35S promoter (von Schaewen et al., 1990). Upon inoculation with G. intraradices, a correlation between leaf invertase activity and root colonization rate was found. Surprisingly, slightly increased (between two- and fourfold) leaf invertase activity stimulated the mycorrhization at early time-points of colonization (3 weeks after inoculation), whereas strongly increased (between 10- and 25-fold) invertase activity in source leaves repressed AM, especially at later time-points (6 weeks after inoculation). As AM fungi are dependent on the carbon supplied by the plant host, the reduced mycorrhization in the latter case might be caused by a diminished carbohydrate level in the roots. In NT 35S::cwINV plants with strongly increased leaf invertase activity the enhanced cleavage of sucrose in source leaves causes a defective sucrose translocation to the sink organs. This results in severe hexose accumulation in the leaves accompanied by downregulation of photosynthesis, chlorosis and growth defects, leading to an undersupply of the root with carbohydrates (see also von Schaewen et al., 1990; Sonnewald et al., 1991). It is highly probable that this insufficient carbon translocation in the plant results in starvation of the AM fungus and therefore reduces fungal growth. This was visible not only in the overall mycorrhization rate, but also in the diminished formation of fungal storage organs (Figure 2f). Moreover, this effect seems to be similar to that shown for NT rolC::ppa plants with defective phloem loading, which was achieved by phloem-specific expression of a pyrophospatase gene from E. coli (Figure S3; Schaarschmidt et al., 2007). These transgenic tobacco plants are characterized by a defective translocation of sucrose to the sink organs resulting in sugar accumulation in source leaves, drastic growth defects and an undersupply of the roots inter alia with hexoses.

In addition, drastic changes in the sink/source relations by increased apoplastic invertase activity and increased accumulation of hexoses can result in a stress response of the plant (Roitsch et al., 2003; Rolland et al., 2006). An elevated defense status of homozygous NT 35S::cwINV plants was previously indicated by transcript accumulation of PR genes and increased tolerance against virus infection (Herbers et al., 1996). In the present study, we detected with increasing apoplastic leaf invertase activity, increased PR transcript accumulations in leaves of heterozygous plants. In the roots, which exhibited no or only slightly enhanced invertase activity, no increased accumulation of PR gene transcripts was found. Thus, transcription of PAR1, PR-1b and PR-Q seemed to be only locally induced by strongly increased leaf invertase activity. It is known that defense-related mechanisms are somehow induced upon AM, most likely to restrict fungal growth to the root cortex and avoid excessive colonization (for reviews, see García-Garrido and Ocampo, 2002; Hause and Fester, 2005). Several studies revealed expression of defense-related genes upon AM (e.g. Salzer et al., 2000; Liu et al., 2003; Gao et al., 2004). However, transcripts of genes associated with plant-defense response often accumulate in a very specific manner and at different stages of AM symbiosis, e.g. the initial phase of AM establishment (Volpin et al., 1994; Liu et al., 2003), at different subcellular sites like arbuscule-containing cells (Blee and Anderson, 1996, 2000) or isoenzyme-specific gene expression as shown for chitinases (Pozo et al., 1998; Salzer et al., 2000). This indicates an induction of specific, defense-related regulatory mechanisms upon AM to control the mutualistic interaction by the plant. Although a higher transcript accumulation of PR genes could not be detected in the respective roots, it cannot be excluded that repression of mycorrhization in NT 35S::cwINV plants with strong invertase induction in the leaf apoplast is at least partially a result of increased hexose accumulation in the source leaves leading to an elevated defense status of the whole plant (Figure 7a). However, such effects on AM fungal growth in the roots by activation of defense-related mechanisms in the leaves is highly speculative. Therefore, diminished mycorrhization in roots of plants with highly increased apoplastic invertase activity might be mainly caused by the undersupply of these roots with carbohydrate.

image

Figure 7.  Proposed model for the regulation of arbuscular mycorrhiza by increased apoplastic invertase activity in leaves of NT 35S::cwINV plants. (a) Transgenic plants with strong invertase expression in leaves are characterized by a severe hexose accumulation in the source leaves, increased transcript levels of PR genes in leaves, indicating increased defense status of the plant, and an undersupply of the root with carbohydrates. These plants showed reduced mycorrhization. (b) Transgenic plants with moderate invertase expression showed in some cases reduced hexose levels in leaves. All of those plants are characterized by reduced contents of defense-related metabolites in roots and increased levels of abscisic acid in roots. These plants showed increased mycorrhization. Hex, hexose content; INV, apoplastic leaf invertase activity; Myc, mycorrhization.

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In contrast to NT 35S::cwINV plants with strongly elevated invertase activity, plants with slightly elevated leaf invertase activity showed no accumulation of PAR1, PR-1b and PR-Q transcripts. This corresponds to the lack of hexose accumulation in the leaves or even to the reduced hexose: sucrose ratio in leaves accompanied by marginal phenotypical symptoms. Analyzing the steady-state level of polar metabolites in wild-type plants and NT 35S::cwINV plants with enhanced mycorrhization, the latter showed reduced levels of chlorogenic acid, scopolin, scopoletin and all identified amines, including e.g. tyramine and octopamin, in roots. Moreover, levels of benzylglucopyranoside and amino acids, including GABA and 5-amino-valeric acid, were reduced in the roots of transgenic plants compared with wild-type. Such metabolites are known to accumulate in response to biotic stress and are thought to enhance plant resistance (Solomon and Oliver, 2001; Cowley and Walters, 2002; Matsuda et al., 2005; Vermerris and Nicholson, 2006). Interestingly, chlorogenic acid isomers, scopolin and scopoletin, accumulated not only in PVY-infected tobacco plants but also in leaves of homozygous NT 35S::cwINV plants: exhibiting in return resistance against PVY (Baumert et al., 2001). Amines and amino acids like amino-butyric acid and amino-valeric acid are suggested to be involved in plant defense, particularly against pathogenic fungi. GABA was shown to accumulate, in addition to some other amino acids, in tomato upon infection with the biotrophic fungi Cladosporium fulvum (Solomon and Oliver, 2001) and is further discussed for a role in plant response to wounding and herbivore attack (Bown et al., 2006). The GABA isomer β-amino-butyric acid (BABA) was found to protect Arabidopsis against Botrytis cinerea and Peronospora parasitica (Zimmerli et al., 2000, 2001), and can induce local and systemic resistance in several pathosystems (Cohen, 2002). Amines, such as octopamine, putrescine and spermidine, accumulated in elicitor-treated potato tubers and in barley upon hypersensitive response to Blumeria graminis hordei (Cowley and Walters, 2002; Matsuda et al., 2005). Moreover, N-(hydroxycinnamoyl)-amines, such as N-feruloyltyramine and 4-N-coumaroyltyramine, appeared to be involved in cell wall fortification in response to Phytophthora infestans attack, and thereby might affect fungal growth in the apoplastic space (Schmidt et al., 1999).

Thus, decreased accumulation of defense-related metabolites might indicate a reduced defense status in roots of those 35S::cwINV plants leading to increased AM fungal colonization of the root. In contrast to the roots, however, leaves with slightly enhanced invertase activity showed minor or non-reproducible metabolic changes. Reduced contents of amines and chlorogenic acid in leaves of some 35S::cwINV plants of group A (experiment I, 3 weeks after inoculation) might be caused by a decreased hexose: sucrose ratio in their leaves. As severely elevated hexose: sucrose ratios in source tissue induce defense reactions by hexose sensing (Herbers et al., 1996), a reduced hexose: sucrose ratio might downregulate or repress such mechanisms.

Most detected reductions in the metabolic steady-state of the analyzed NT 35S::cwINV plants, particularly the reduced levels of defense-related compounds in the root, seemed not to be the result of increased mycorrhization, as one could assume. Because the mycorrhization levels of both experiments differ drastically, changes in the metabolite profile caused by increased colonization would even be obvious by comparing the wild-type plants of both experiments. However, corresponding changes were absent, particularly for amino acids, phenolic compounds or amines (see also Figure S6). This is supported by previous studies on Medicago truncatula, in which phenolic compounds, amines and amino acids were not reduced but were sometimes even slightly elevated upon inoculation with G. intraradices (Lohse et al., 2005). Moreover, G. intraradices-colonized barley showed an at least transient accumulation of hydroxycinnamic acid amids (Peipp et al., 1997). These findings further support our hypothesis that downregulation of defense-related mechanisms might enhance mycorrhization of transgenic plants with slightly elevated invertase activity in the leaf apoplast. In addition to reduced contents of such defense-related metabolites, we detected in NT 35S::cwINV plants exhibiting enhanced mycorrhization increased ABA levels in the roots. Reports concerning ABA levels in mycorrhizal wild-type plants give contrary results as some describe an increase upon AM in roots and leaves (Danneberg et al., 1992; Bothe et al., 1994; Meixner et al., 2005), whereas others found a decrease in leaves (Allen et al., 1982). However, ABA is known to determine basal susceptibility in plant–microbe interaction – both in pathogenic and in symbiotic ones. Tomato mutants with reduced ABA levels showed not only higher resistance upon infection with the necrotrophic fungus B. cinerea (Audenaert et al., 2002), but are also characterized by a reduced AM formation (Herrera et al., 2006). In both systems, application of exogenous ABA restored susceptibility of the mutant and increased the susceptibility of the wild-type.

Thus, increased AM formation in NT 35S::cwINV plants with slightly enhanced leaf invertase activity might result from reduced levels of defense-related metabolites, such as amines, phenolics and some amino acids, and additionally increased ABA contents in the root, which could, somehow, be regulated by the carbohydrate status of the plant (Figure 7b).

Summarizing the presented data, we found a crucial function of invertase activity in the leaf apoplast and the sugar status of source leaves in regulating the AM symbiosis. The symbiotic interaction between root-colonizing AM fungi and plants seemed to be controlled inter alia by the carbohydrate status of the leaves affecting carbohydrate supply, hormone status, PR gene transcripts and stress-related metabolites in the root. By this, the AM formation can either be enhanced or decreased, independently of the apoplastic invertase activity in the root.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant material and growth conditions

Wild-type tobacco plants (N. tabacum cv. Samsun NN) were obtained from Vereinigte Saatzuchten eG (http://www.vs-ebstorf.de) and germinated on solid MS medium (Duchefa, http://www.duchefa.com). Seeds of a heterozygous line of NT 35S::cwINV (von Schaewen et al., 1990), kindly provided by U. Sonnewald (University of Erlangen, http://www.uni-erlangen.org), were sown on solid MS medium containing 50 mg l−1 kanamycin A (Duchefa) for selection. All plants were cultivated in a growth chamber with 16-h light (250 μmol photons m−2 sec−1; Philips Powerstar HQI 250/D lamps; Philips, http://www.philips.com) and 8-h dark at 25°C and 50% relative humidity. After 3 weeks on solid medium, plants were transferred into pots filled with expanded clay of 2–5-mm particle size (Original Lamstedt Ton; Fibo ExClay, http://www.fiboexclay.de). Plants were watered with distilled water and fertilized twice per week with 10 ml 10× Long Ashton (20% phosphate) (Hewitt, 1966). After inoculation with the AM fungus, plants were grown under the conditions described above but fertilized with 25 ml 10× Long Ashton (20% phosphate) twice per week. Roots and middle-aged leaves were harvested at the end of the light period. Except the material used for staining, samples were immediately frozen in liquid nitrogen and stored at −80°C until use.

Inoculation with G. intraradices, staining of fungal structures and determination of fungal rRNA

For inoculation, the AM fungus G. intraradices Schenk & Smith isolate 49 (Maier et al., 1995) was used after enrichment by previous co-cultivation with leek (Allium porrum cv. Elefant). Six-week-old tobacco plants were inoculated by careful removal of the previous substrate and transfer to new pots filled with expanded clay containing G. intraradices inoculum freshly harvested from mycorrhizal leek plants. Non-mycorrhizal plants were transferred in the same way to pure expanded clay. For the estimation of G. intraradices colonization a representative cross section of each root system was taken. The mycorrhizal structures in the root pieces were stained according to the method described by Vierheilig et al. (1998) using 5% (v/v) ink (Sheaffer Skrip jet black, Sheaffer, http://www.sheaffer.com) in 2% (v/v) acetic acid and analyzed using a stereomicroscope. Micrographs of ink-stained roots were taken using a Zeiss ‘Axioplan’ microscope (Zeiss, http://www.zeiss.com) equipped with a video camera (Fujix Digital Camera HC-300Z; Fuji Photo Film, http://www.fujifilm.com) and were processed through PHOTOSHOP 7.0 (Adobe Systems, http://www.adobe.com).

The level of G. intraradices-specific rRNA in roots was quantified by real-time RT-PCR. Total RNA of mycorrhizal roots was isolated using the Qiagen RNeasy Plant Mini Kit (Qiagen, http://www1.qiagen.com) including a DNase-digestion (RNase-free DNase Set; Qiagen). First-strand cDNA synthesis of 1 μg RNA in a final volume of 20 μl was performed with M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant (Promega, http://www.promega.com) according to the supplier’s protocol using random hexamer primer. For real-time PCR, 4.5 μl of 1:9 diluted cDNA (25 ng reverse transcribed total RNA) were mixed with 2× TaqMan Master Mix (Applied Biosystems, http://www.appliedbiosystems.com) and 20× TaqMan probe and primers (Assays-by-Design; Applied Biosystems) in a final volume of 10 μl in three independent replicates. TaqMan probes and primers for G. intraradices-specific rRNA were used as described previously (Isayenkov et al., 2004). As an internal control for cDNA-synthesis, transcript levels of N. tabacum Ubiquitin were measured using the following probe and primers: probe, 5′-GGAAGCAGCTCGAGGAT-3′; forward primer, 5′-CCAGCAGAGGTTGATCTTTGC-3′; reverse primer, 5′-AAGGACCTTGGCTGACTACAAT ATC-3′ (Custom Taqman Gene Expression Assay, Assays-by-Design; Applied Biosystems). Real-time PCR was performed using the Mx 3005P QPCR system (Stratagene, http://www.stratagene.com) according to the Assays-by-Design protocol (Applied Biosystems). Data were evaluated with the mxpro software (Stratagene) and calculated by the comparative Ct method.

Determination of invertase activity and soluble sugar contents

Invertase activity was measured as described (Schaarschmidt et al., 2004). Determination of soluble sugar contents was performed photometrically by a coupled enzymatic assay as described previously (Schaarschmidt et al., 2004).

Determination of total chlorophyll content and phenotypic analysis

For chlorophyll determination, leaf material was homogenized in liquid nitrogen. Of each sample, 50 mg was extracted twice with 1.5 ml of absolute methanol. The supernatant (in total 3 ml) was collected and diluted 1:1 with methanol. The total chlorophyll content in 1 ml diluted extract was measured spectrophotometrically (DU 640 Beckmann Spectrophotometer; Beckmann Instruments, http://www.beckmancoulter.com) against methanol at a wavelength of 664.5 nm, showing adsorption maxima of the extract. Determination was carried out in two independent replicates.

As phenotypical markers of growth reduction, stem length, total number of fully developed leaves and length of the youngest and oldest counted leaf, as well as length of the largest leaf, were determined for each plant. Leaf length corresponded with leaf width and total leaf area.

Analysis of transcript accumulation of PR genes

Total RNA isolation and cDNA synthesis of pooled samples of roots and middle-aged leaves of at least three parallel plants was performed as described above for the determination of fungal rRNA, but with oligo dT (T19) primer instead of random hexamer primer for cDNA synthesis. After RT, PCR was performed with 0.5 or 3 μl template in a total volume of 50 μl using GoTaq DNA Polymerase (Promega), 5× Green GoTaq Flexi Buffer (Promega), 2 mm MgCl2, 0.2 mm dNTP and 0.5 μm each of forward and reverse primer. Primers were used as follows: PAR1, forward primer, 5′−GAAGCGTTGCGTGTTAGAG-3′, reverse primer, 5′-CACTGGTCGGTTTCAATCC-3′; PR-Q, forward primer, 5′-TTGGCACAAGGCATTGGTTC-3′, reverse primer, 5′-CTTGTTGTCCTGTGGTGTCATC-3′; PR-1b, forward primer, 5′-GTAGGCGTGGAACCATTAAC-3′, reverse primer, 5′-GCACTTAACCCTAGCACATC-3′; Ubiquitin, forward primer, 5′-TGACTGGGAAGACCATCAC-3′, reverse primer, 5′-TAGAAACCACCACGGAGAC-3′. PCR conditions were for all primers: 2 min at 95°C, followed by 25 cycles of 30 sec at 95°C, 30 sec at 56°C, 40 sec at 72°C, and additional 2 min at 72°C. After the reaction, 20 μl of the assay was separated on a 1% (w/v) agarose gel stained with ethidium bromide.

Steady-state analysis of polar metabolites

After homogenization in liquid nitrogen, 120 ± 5 mg of leaf or root material from mycorrhizal plants were extracted with 300 μl pre-cooled (−20°C) absolute methanol. To each sample, 30 μl of a methylnonadecanoate stock solution (2 mg ml−1 methanol) (Sigma-Aldrich, http://www.sigmaaldrich.com) and 30 μl of a ribitol stock solution (0.2 mg ml−1 methanol) (Sigma-Aldrich) were added. After shaking at 70°C for 15 min, 200 μl chloroform was added to the samples, which were shaken at 37°C for an additional 5 min. Then 400 μl H2O was added. After vortexing, centrifugation was performed at 16 000 g for 5 min at room temperature (20°C). For back-up and validation purposes, 80 and 160-μl aliquots of the upper polar phase were dried by vacuum centrifugation (Concentrator 5301; Eppendorf, http://www.eppendorf.com). Chemical derivatization (Roessner et al., 2000; Fiehn et al., 2000) and GC-TOF-MS metabolite profiling analysis (Wagner et al., 2003) was performed essentially as described previously. Metabolites were identified using the NIST05 mass spectral search and comparison software (National Institute of Standards and Technology; http://www.nist.gov/srd/mslist.htm) and the mass spectral and retention index (RI) collection (Schauer et al., 2005) of the Golm Metabolome Database (GMD; Kopka et al., 2005). Metabolites are characterized by chemical abstracts system (CAS) identifiers and compound codes issued by the Kyoto Encyclopedia of Genes and Genomes (KEGG; Kanehisa et al., 2004). RIs represent Kovàts indices (Kovàts, 1958) calculated form standard additions to each chromatogram of a mixture of C12, C15, C19, C22, C32, C36n-alkanes. Metabolites were quantified after mass spectral deconvolution (chromatof software version 1.00, Pegasus driver 1.61; LECO, http://www.leco.com) of at least two mass fragments of metabolite derivatives, here called analytes. Peak height, representing an arbitrary mass spectral ion current of each mass fragment, was normalized using the quantity of the sample FW of the ribitol analyte for internal standardization of volume variations.

Independent component analysis and statistical assessment of GC-TOF-MS fingerprints

Independent component analysis (ICA; Scholz et al., 2004) was applied to the metabolite fingerprints. Data were normalized by calculation of response ratios using the median of each row as denominator and subsequent logarithmic transformation. Missing value substitution was as described earlier (Scholz et al., 2005). Statistical testing was performed using the Student’s t-test. Logarithmic transformation of response ratios approximated the required Gaussian normal distribution of metabolite profiling data.

Determination of ABA

The content of ABA in roots and leaf material was determined as described previously (Meixner et al., 2005) using ca. 100 mg FW of plant material per sample. During extraction 50 ng (d6)-ABA (Plant Biotechnology Institute, National Research Council of Canada, http://pbi-ibp.nrc-cnrc.gc.ca) were added to each sample. For GC-MS analysis the sample was methylated with freshly prepared diazomethane (Cohen, 1984). GC-MS analysis was carried out on a Varian Saturn 2100 ion-trap mass spectrometer using electron impact ionization at 70 eV, connected to a Varian CP-3900 gas chromatograph equipped with a CP-8400 autosampler (Varian, http://www.varianinc.com). For the analysis 2.5 μl of the methylated sample dissolved in 20 μl ethyl acetate was injected onto a Phenomenex (http://www.phenomenex.com) ZB-5 column (30 m × 0.25 mm × 0.25 μm) using He carrier gas at 1 ml min−1. Injector temperature was 250°C and the temperature program was 60°C for 1 min, followed by an increase of 25°C min−1 to 180°C, 5°C min−1 to 250°C, 25°C min−1 to 280°C, and then 5 min isothermically at 280°C. Transfer line temperature was 280°C and the trap temperature 200°C. For higher sensitivity, the μSIS mode (Varian Manual; Wells and Huston, 1995) was used. For the determination of ABA the ions of the methylated substance at m/z 190/194 were monitored and the endogenous hormone concentrations were calculated by the principles of isotope dilution (Cohen et al., 1986).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We gratefully acknowledge Prof. Uwe Sonnewald for providing seeds of transgenic N. tabacum 35S::cwINV plants. Furthermore, we thank Prof. Dieter Strack for critically reading the manuscript. Alexander Erban is acknowledged for his helpful assistance with the metabolite analysis and annotation. We also want to thank Christine Kaufmann for creating the tobacco drawing.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Figure S1. Soluble sugar contents in mycorrhizal invertase-overexpressing plants. (a) The sum of the glucose and fructose contents in roots and leaves of wild-type and NT 35S::cwINV plants. (b) The sucrose content in these plants. The experimental set-up and the classification of heterozygous NT 35S::cwINV plants by their leaf invertase activity is described in Figure 1. Data are given as mean values + SD of 3 to 8 plants per group. Different letters designate statistically different sugar contents (ANOVA with Tukey HSD test, P<0.05). Figure S2. Invertase activity, soluble sugar contents and growth parameters of non-mycorrhizal invertase-overexpressing plants. The plants were cultivated in parallel to the G. intraradices-inoculated plants shown in Figure 1 and Figure S1. Data are exemplarily presented for the early time-point of experiment I (3 weeks) and for the late time-point of experiment II (6 weeks). (a) Invertase activity in roots and leaves of non-mycorrhizal wild-type and NT 35S::cwINV plants. (b) Ratio of the sum of glucose and fructose to the sucrose content of these plants. (c) The sum of the glucose and fructose contents in roots and leaves of wild-type and NT 35S::cwINV plants. (d) The sucrose content in these plants. (b) Length of stem, number of full-developed leaves and length of the largest leaf, used as indicator for leaf size, of wild-type and NT 35S::cwINV plants at the 6-week-time-point. All plants were grouped by their leaf invertase activity as described in Figure 1. Data are given as mean values + SD of 3 to 9 plants per group and are tested with one-way ANOVA followed by Tukey HSD test. P<0.05. Means sharing the same letters are not significantly different. Figure S3. Formation of fungal structures in invertase-overexpressing plants. (a,b) Wild-type plant. (c,d) NT 35S::cwINV plant of group A, characterized by slightly enhanced invertase activity in leaves (below 50 pkat mg-1 [protein]). (e) NT 35S::cwINV plant of group C with strongly enhanced invertase activity in leaves (more than 100 pkat mg-1 [protein]). (f) Transgenic NT rolC::PPa plant with pronounced phenotype, characterized by a general undersupply of the root (Lerchl et al., 1995; Schaarschmidt et al., 2007). All plants were cultivated in parallel and inoculated with G. intraradices 6 weeks after sowing. Roots were harvested 4.5 weeks after inoculation. Cross sections of 140 μm thickness of G. intraradices-colonized roots were stained with two fluorescent labeled wheat germ agglutinins (WGAs). The fluorescence of WGA-TRITIC showing high affinity to arbuscules and hyphae is given in red, the fluorescence of WGA-Alexa Fluor 488, which additionally labeled fungal vesicles (V), in green. In the shown overlay, structures labeled by both fluorescent WGAs as arbuscules (A) and hyphae (H) appear in yellow. The formation of mycorrhizal structures was analyzed with a confocal laser scanning microscope (LSM 510 Meta, Zeiss, Jena, Germany) using the 488 nm (Alexa Fluor 488) and 543 nm (TRITC) laser lines for excitation. Series of optical sections (z-series) were acquired by scanning 10 sections with a distance of 0.2 μm on the z-axis; z-series projections were done with the LSM Image Examiner software (Zeiss). Bars = 50 μm. Note the high colonization of the root cortex in NT 35S::cwINV with slightly increased invertase activity (c) compared to wild-type (a) and the formation of well-developed arbuscules (b,d). In contrast, less fungal structures including mostly small arbuscules were found in the roots of NT 35S::cwINV plants with strongly increased leaf invertase activity (e). This corresponds to the colonization of NT rolC::PPa plants (f), expressing the E. coli pyrophosphate gene ppa with a phloem-specific promoter. In these plants the PPi-dependent phloem-loading is defective resulting in growth reduction of the plant and an undersupply of the root. Figure S4. Indole-3-acetic acid (IAA) content in wild-type plants and NT 35S::cwINV plants showing increased mycorrhization. The content of total, free and conjugated IAA in roots and leaves of mycorrhizal wild-type and 35S::cwINV tobacco plants of group A, characterized by slightly elevated leaf invertase activity (below 50 pkat mg-1 [protein]), 3 weeks after inoculation with G. intraradices are shown. Determination of IAA was performed as described previously (Fitze et al., 2005). Data of two independent experiments (experiment I and II; see Figure 1) are given each as mean value + SD of 3 to 5 plants. In total 8 wild-type and 9 transgenic plants were analyzed. Data of the transgenic plants were compared to the wild-type by the Student t test. *P<0.05, **P<0.002. Figure S5. Steady-state levels of selected polar metabolites with increased levels in NT 35S::cwINV plants showing increased mycorrhization compared to wild-type plants. Contents of phosphoric acid, several phosphates, raffinose, carbodiimide, and a novel amine (provided with its mass spectra) in roots and leaves are shown. For each component the identified mass fragment with the highest response from the metabolite profile of Table S1 was chosen. The data are given for root and leaf samples of wild-type (wt) and 35S::cwINV plants of group A (INV) with slightly enhanced invertase activity in leaves (below 50 pkat mg-1 [protein]) of two independent experiments (exp I and exp II; see Figure 1). For each experiment the mean values + SD of 3 to 5 wild-type or transgenic plants are shown. The change of pool size for roots is given as ratio of transgenic and wild-type root levels for each experiment. Note particularly the increased levels of the shown phosphorous- and nitrogen-containing compounds in roots and leaves of 35S::cwINV plants of group A compared to wild-type plants. Figure S6. Steady-state levels of selected polar metabolites with decreased levels in NT 35S::cwINV plants showing increased mycorrhization compared to wild-type plants. Contents of 5-amino-valeric acid, 4-amino-butyric acid, benzylglucopyranoside, the identified amines, chlorogenic acid isomers, scopolin, and scopoletin in roots and leaves are shown. For each component the identified mass fragment with the highest response from the metabolite profile of Table S1 was chosen. The data are given for root and leaf samples of wild-type (wt) and 35S::cwINV plants of group A (INV), characterized by slightly elevated invertase activity in leaves (below 50 pkat mg-1 [protein]), of two independent experiments (exp I and exp II; see Figure 1). For each experiment the mean values + SD of 3 to 5 wild-type or transgenic plants are shown. The change of pool size for roots is given as ratio of transgenic and wild-type root levels for each experiment. Note the decreased levels of those defense-related compounds in the roots of 35S::cwINV plants of group A compared to wild-type plants. Table S1. Metabolite profile of steady-state pool sizes of polar metabolites from wild-type plants and apoplastic invertase-overexpressing plants with increased mycorrhization. These data represent the subset of identified mass fragments from the metabolite fingerprint. Metabolites are grouped according to chemical class and characterized by sum formula, KEGG and CAS identifier. Analytes used to monitor metabolites in GC-TOF-MS profiles are characterized by chemical derivatization and CAS as well as MPIMP identifier (MPIMP-ID; Kopka et al., 2005). Validation of identification is documented by deviation of expected from found RI, mass spectral match factor, frequency of occurrence and choice of selective mass fragments. Furthermore average and standard deviation of each replicate sample group and relative changes of pool sized comparing 35S::cwINV of group A (INV) to wild-type (wt) plants are given. Statistical testing was performed as described in materials and methods. In total 8 wild-type and 9 transgenic plants were analyzed. Significant changes (p<0.05) between wild-type and transgenic plants are marked in bold, increase in the transgenic in red and decrease in green.

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TPJ_3150_sm_FigS1.tif1147KSupporting info item
TPJ_3150_sm_FigS2.tif1388KSupporting info item
TPJ_3150_sm_FigS3.tif4231KSupporting info item
TPJ_3150_sm_FigS4.tif644KSupporting info item
TPJ_3150_sm_FigS5.tif4888KSupporting info item
TPJ_3150_sm_FigS6.tif3184KSupporting info item
TPJ_3150_sm_TableS1.xls309KSupporting info item

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