Regulation of arbuscular mycorrhization by carbon. The symbiotic interaction cannot be improved by increased carbon availability accomplished by root-specifically enhanced invertase activity

Abstract

The mutualistic interaction in arbuscular mycorrhiza (AM) is characterized by an exchange of mineral nutrients and carbon. The major benefit of AM, which is the supply of phosphate to the plant, and the stimulation of mycorrhization by low phosphate fertilization has been well studied. However, less is known about the regulatory function of carbon availability on AM formation. Here the effect of enhanced levels of hexoses in the root, the main form of carbohydrate used by the fungus, on AM formation was analyzed. Modulation of the root carbohydrate status was performed by expressing genes encoding a yeast (Saccharomyces cerevisiae)-derived invertase, which was directed to different subcellular locations. Using tobacco (Nicotiana tabacum) alcc::wINV plants, the yeast invertase was induced in the whole root system or in root parts. Despite increased hexose levels in these roots, we did not detect any effect on the colonization with Glomus intraradices analyzed by assessment of fungal structures and the level of fungus-specific palmitvaccenic acid, indicative for the fungal carbon supply, or the plant phosphate content. Roots of Medicago truncatula, transformed to express genes encoding an apoplast-, cytosol-, or vacuolar-located yeast-derived invertase, had increased hexose-to-sucrose ratios compared to beta-glucuronidase-transformed roots. However, transformations with the invertase genes did not affect mycorrhization. These data suggest the carbohydrate supply in AM cannot be improved by root-specifically increased hexose levels, implying that under normal conditions sufficient carbon is available in mycorrhizal roots. In contrast, tobacco rolC::ppa plants with defective phloem loading and tobacco pyk10::InvInh plants with decreased acid invertase activity in roots exhibited a diminished mycorrhization.

Figure 1.

Induction of an apoplast-located invertase in root parts of transgenic tobacco plants and effects on mycorrhization. A to C, NT alc∷cwINV plants were cultivated using the split-root system where both root parts were either inoculated with G. intraradices 6 weeks after sowing (myc) or left as controls without inoculation (non-myc). To induce expression from the chimeric invertase gene, a defined root from the split-root plants was drenched with 100 mL of 0.05% (v/v) aqueous acetaldehyde solution (Aa). The control root was treated with water (H₂O). Soil drenching was performed in weekly intervals 0, 7, and 14 d after inoculation as indicated by arrows. A, Invertase activity of the root parts. B, Ratio of the Glc and Fru contents to the Suc content of the root parts. C, Degree of mycorrhization of the root parts of single plants. D, Content of C16:1Δ¹¹ and C16:0 in single root parts of mycorrhizal plants 6 and 7 weeks after inoculation. Plants harvested 6 weeks after inoculation are described above (A–C), plants harvested at week 7 were cultivated and inoculated in the same way but drenched with acetaldehyde 21, 28, and 35 d after inoculation. Data are expressed in A and B as means ± sd. (n = 3). In C, data of single root parts and the mean values of the three parallel plants are given.

Figure 2.

Formation of fungal structures in water- and acetaldehyde-treated wild-type and alc∷cwINV roots. Cross sections of 140 μm thickness of G. intraradices-colonized roots were stained with two fluorescent-labeled WGAs. The fluorescence of WGA-TRITC showing high affinity to arbuscules and hyphae is shown in red, and the fluorescence of WGA-Alexa Fluor 488, which additionally labeled fungal vesicles (v), is in green. In the overlay, structures labeled by both fluorescent WGAs as arbuscules (a) and hyphae (h) appear in yellow. Plants were harvested 6 weeks after inoculation. Drenching of the whole root system with 100 mL 0.05% (v/v) acetaldehyde for root-specific induction of apoplastic invertase was performed five times at weekly intervals starting with the time point of inoculation (+Aa). Control plants were water treated (+H₂O). Bars = 50 μm.

Figure 3.

Inorganic phosphate content of roots and leaves of water- and acetaldehyde-treated wild-type and alc∷cwINV plants. Six-week-old plants were either inoculated with G. intraradices (myc) or left without inoculation (non-myc). Invertase induction in the whole root system was performed as described in Figure 1 for root part-specific induction. Data are presented as mean values + sd (nonmycorrhizal plants: n = 4; mycorrhizal plants: n = 6) and are tested with multiple t tests, including Bonferroni correction. P < 0.05. Means sharing the same letters are not significantly different.

Figure 4.

Biomass analysis of nonmycorrhizal and mycorrhizal wild-type and transgenic tobacco plants exhibiting increased apoplastic invertase activity in the root. A, Root-to-shoot ratio of the fresh weight of 8-week-old nonmycorrhizal wild-type and alc∷cwINV plants. These plants were soil drenched twice with 100 mL 0.05% (v/v) acetaldehyde (Aa) or water (H₂O) 1 and 2 weeks before harvest. Mean values of two independent experiments + sd are given (wild type: n = 22; alc∷cwINV: mean values of three independent lines with each n = 30). B, Root-to-shoot ratio of the fresh weight of 13-week-old wild-type and alc∷cwINV plants inoculated with G. intraradices 7 weeks before harvest. Acetaldehyde (Aa) or water (H₂O) was applied as described for A, but was carried out weekly four times starting 7 d after inoculation. Results are the mean + sd (wild type: n = 24; alc∷cwINV: mean values of three independent lines with each n = 16).

Figure 5.

Analysis of root-transformed M. truncatula plants expressing genes encoding yeast-derived invertase directed to different subcellular locations. A, Ratio of the sum of Glc and Fru to the Suc content of the transformed roots. B, Relative levels of G. intraradices rRNA and of transcripts of the mycorrhiza-induced phosphate transporter MtPT4 in mycorrhizal roots. Transcript and rRNA levels of roots transformed with the GUS construct were set to 1. C and D, Inorganic phosphate content of roots (C) and leaves (D). E, Content of C16:1Δ¹¹ and C16:0 in mycorrhizal roots. A. rhizogenes-mediated root transformation was performed using constructs containing the gene coding for yeast invertase, which is either translocated to the apoplast (cwINV), the cytosol (cytINV), or the vacuole (vacINV), or the gene coding for GUS, all expressed under control of the 35S promoter. Five weeks after transformation, plants were inoculated with G. intraradices (myc) or left without inoculation (non-myc) and were harvested 5 weeks later. All results are presented as means + sd (nonmycorrhizal plants: n = 4; mycorrhizal plants: n = 6). Data of nonmycorrhizal and mycorrhizal plants root transformed with plasmids coding for the different located invertases were compared to nonmycorrhizal and mycorrhizal plants root transformed with the GUS construct, respectively, using the Student's t test. *P < 0.05, **P < 0.01.

Figure 6.

Analysis of transgenic tobacco plants with phloem-specific expression of a pyrophosphatase. A, Phenotypes of 9-week-old nonmycorrhizal wild-type (left-most position) and rolC∷ppa plants. Heterozygous NT rolC∷ppa plants were classified by their growth reduction into three groups: large, intermediate, and small (from left to right; see different shadings). B, Biomass analysis. Ratio of root fresh weight to shoot fresh weight of nonmycorrhizal and mycorrhizal wild-type and rolC∷ppa plants 3 and 5 weeks after inoculation. Wild-type and rolC∷ppa plants were inoculated with G. intraradices 6 weeks after sowing (myc) or left without inoculation (non-myc). C, Root-to-shoot ratio of the sum of Glc and Fru in the mycorrhizal plants. Ratios of NT rolC∷ppa plants comprehend data of large, intermediate, and small plants. D, Mycorrhization degree of the inoculated plants. Data in B, C, and D are given as means + sd (n ≥ 6). For each developmental stage, the fresh weight and hexose ratios of NT rolC∷ppa plants were compared to wild-type plants using the Student's t test. *P < 0.05, **P < 0.01. E and F, Ink-stained fungal structures in a wild-type (E) and an intermediate rolC∷ppa plant (F) 4 weeks after inoculation. Bars represent 100 μm.

Figure 7.

Analysis of transgenic tobacco plants with root-specific expression of an invertase inhibitor. A and B, Cell wall (A) and vacuolar (B) invertase activity in roots of wild-type SR1 plants and NT pyk10∷InvInh plants of two independent lines (98-1-10 and 98-4-1) 3.5 and 5 weeks after inoculation with G. intraradices. C, Glc and Fru content of the roots. D, Ratio of the Glc and Fru contents to the Suc content of the roots. E, Degree of mycorrhization. To allow statistical analysis, the degree of mycorrhization in percent of the root length was determined for every root system in 50 to 100 root pieces of each 1 cm length. Plants were in two independent experiments inoculated with G. intraradices either 2.5 weeks after sowing and harvested 3.5 weeks later or inoculated 4 weeks after sowing and harvested 5 weeks later. Data are presented as mean values + sd (at 3.5 weeks: n = 5; at 5 weeks: n = 3). The data from the transgenic lines were pairwise compared to the wild type by the Student's t test. *P < 0.05, **P < 0.01. F, Ink-stained fungal structures in a wild-type and a NT pyk10∷InvInh plant of line 98-1-10, each 5 weeks after inoculation. Bars represent 100 μm.

Figure 8.

Biomass analysis of NT pyk10∷InvInh plants. Root-to-shoot ratio of the fresh weight of 6-week-old nonmycorrhizal wild-type SR1 plants and plants of two independent NT pyk10∷InvInh lines (98-1-10 and 98-4-1). Mean values of +sd are given (n ≥ 33).